Elevation of oil levels in plants

ABSTRACT

This present invention provides a method for increasing oil levels in corn kernel tissue by expression of an HOI001 GBSS allele. The present invention also provides isolated nucleic acid molecules encoding a HOI001 GBSS polypeptide.

This application claims the benefit of the filing date of theProvisional Application U.S. Ser. No. 60/483,491, filed Jun. 27, 2003,which is incorporated herein by reference.

The present invention relates to the fields of nucleic acid chemistryand agricultural biotechnology. In particular, the present invention isdirected at the identification of nucleic acids that encode proteinsuseful for increasing oil levels in maize plants and creating maizeplants that include such nucleic acids.

Plants are a major source of oils for feed, food, and industrial uses.While tissues of most plant species contain little oil, the cultivationof certain plant types, over many acres, permit large quantities ofplant oils to be produced. If the oil content of these plants could beincreased, then plant oils could be produced more efficiently. Forexample, the normal oil content of yellow #2, dent corn is about 4%. Ifthe oil content of corn could be increased to 8% or even 12%, withoutsignificantly affecting yield, the same amount of oil could be producedfrom half or even one-third the number of acres.

Currently, levels of oil in oilseed crops have increased incrementallyby traditional breeding and selection methods. There exist fewreferences to transgenic plants with increased levels of oil. Incontrast, increases in the proportions of some strategic fatty acidshave been achieved by the introduction or manipulation of various plantfatty acid biosynthesis genes in oilseeds. For instance, Voelker et al.,Science, 257:72-74 (1992), demonstrated that expression in Brassicaceaeof a medium chain fatty acyl-ACP thioesterase from California Bay,increased the lauric acid (12:0) content. Hitz et al., Proc. 9 ^(th)International Cambridge Rapeseed Congress UK, pp 470-472 (1995)increased proportions of oleic acid in Glycine max by co-suppressionusing a sense construct encoding a plant microsomal FAD-2 (Δ12)desaturase. Although the use of these plant transgenes resulted in anincreased production of lauric acid in canola and altered proportions ofoleic acid in soy, there was no evidence of increased total fatty acidcontent, or increased oil yield in these transgenics.

Certain workers have attempted to increase or modulate the oil contentof plants by manipulation of oil biosynthetic pathway genes. Forexample, U.S. Pat. No. 6,268,550 to Gengenbach et al. provides maizeacetyl CoA carboxylase nucleic acids for altering the oil content ofplants. Additionally, U.S. Pat. No. 5,925,805 to Ohlrogge et al.provides an Arabidopsis acetyl CoA carboxylase gene that can be used toincrease the oil content of plants. However, the synthesis of fattyacids requires the coordinated activity of many enzymes, none of whichwhen solely upregulated has been found to substantially increase oilcontent.

A need therefore exists for an improved method to alter the oil contentof plants, and in particular to increase the oil content of plants andseeds.

In addition to oil, starch from maize is also agriculturally andcommercially significant. Starch comprises a major component of animalfeed and human food. Starch is also used industrially in the productionof paper, textiles, plastics, and adhesives, as well as providing theraw material for some bioreactors.

In higher plants, the starch consists of linear chain and branched chainglucans known as amylose and amylopectin, respectively. Starch withvarious amounts of amylose and amylopectin are found in differentplants. Typically, maize starch contains approximately 25% amylose, theremainder being amylopectin. Amylopectin contains short chains and longchains, the short chains ranging from 5-30 glucose units and the longchains ranging from 30-100 glucose units, or more. The ratio of amyloseto amylopectin, as well as the distribution of short to long chains inthe amylopectin fraction, affect the physical properties of starch,(e.g., thermal stabilization, retrogradation, and viscosity).

The WAXY locus of maize determines the amylose content in pollen and inkernel endosperm, (Shure et al., Cell, 35(1):225-233 (1983)), resultingin starch having unique properties. Most mutations in the WAXY locus ofmaize, which encodes granule bound starch synthase (GBSS), result in anopaque endosperm of smooth, firm non-corneous starch comprising mostlyamylopectin and a reduced amount of amylose in the endosperm, pollen andembryo sac (“WAXY phenotype”) (see, Okagaki and Wessler, Genetics,120(4):1137-1143 (1988)). When no functioning GBSS is synthesized in thehomozygous WAXY mutant, it also lacks amylose (Echt and Schwartz,Genetics, 99:275-284 (1981)).

Additionally, classic, recessive WAXY has a small (approximately 0.5%increase) effect on percent oil in the kernel when compared to yellow #2corn (Pfahler and Linskens, Theoretical and Applied Genetics, 41(1):2-4(1971)). In comparison, the inbred line HOI001, a dominant WAXY mutantinbred described in U.S. Patent Publication No. 20030172416, hereinincorporated by reference, has whole kernel oil concentrations greaterthan four times that of yellow #2 corn.

SUMMARY OF THE INVENTION

The present invention describes and provides isolated nucleic acidmolecules encoding an HOI001 GBSS polypeptide. In addition, thisinvention relates to nucleic acid molecules that are complementary tothe nucleic acid molecule encoding an HOI001 GBSS polypeptide. Inaddition, this invention relates to expression cassettes comprisingthese nucleic acid molecules. In addition, this invention relates totransgenic maize plants containing these expression cassettes. Inaddition, this invention relates to the seeds of these transgenic maizeplants. This invention further relates to the oil and animal feedobtained from the seeds of these transgenic maize plants.

In another embodiment, the present invention relates to a recombinantDNA construct, associated with increased oil production in plants,comprising a nucleic acid molecule encoding an HOI001 GBSS polypeptideoperably linked to a promoter, which is functional in a plant cell.

The present invention describes and provides a method of increasing oilin a maize plant by expression of an HOI001 GBSS gene. This inventionfurther provides a method of altering the kernel composition in a cornplant by expression of an HOI001 GBSS gene. This invention furtherdescribes and provides sequences of an HOI001 GBSS gene from Zea mays.This invention further provides vector constructs for planttransformation and tissue-specific expression of an HOI001 GBSS gene.This invention further provides maize plants transformed with the GBSSgene with higher oil levels when compared to plants with the same orsimilar genetic background, but not containing the inserted HOI001 GBSSgene. This invention further provides seeds from these maize plants.This invention further provides for kernels from maize plantstransformed with the HOI001 GBSS gene containing a higher level of oilwhen compared to kernels from corn plants with the same or similargenetic background, but not containing the inserted HOI001 GBSS gene.This invention also provides oil and animal feed produced from theseseeds and kernels.

The present invention further provides a method of marker-assistedbreeding useful in breeding higher oil levels in maize.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleic acid sequence alignment of the granule boundstarch synthase gene isolated from HOI001 (HOI001 GBSS, pMON72506) [SEQID NO: 1] compared to the granule bound starch synthase (GBSS) gene frominbred LH59 (pMON72510), and published sequence of the GBSS genedescribed in Shure et al., supra, (X03935). For additional comparison,the coding sequence for the published GBSS gene is given (CDS22509).

FIG. 2 shows the alignment of the corresponding predicted amino acidsequences from the GBSS gene isolated from HOI001 (HOI001 GBSS frompMON72506) [SEQ ID NO: 3], and the GBSS gene described in Shure et al.,supra, [SEQ ID NO: 4], respectively.

FIG. 3 shows the alignment of the corresponding predicted amino acidsequences from the Zea mays GBSS gene isolated from inbred LH59 [SEQ IDNO: 10], and the Zea mays granule bound starch synthase gene describedin Shure et al., supra, respectively.

FIG. 4 depicts a plasmid map of pMON72506.

FIG. 5 depicts a plasmid map of pMON72510.

FIGS. 6A and 6B graphically depict the difference in oil levels fromkernels of plants transformed with pMON72506 containing the GBSS fromHOI001 (SEQ ID NO: 1, 6A) and pMON72510 containing the GBSS from LH59(SEQ ID NO: 8, 6B). Gene positive and gene negative kernels are comparedfrom each event. Only events with statistically significant changes inoil (14 of 29) are shown in 6A.

FIG. 7 depicts a plasmid map of pMON81464.

FIG. 8 depicts a plasmid map of pMON68298.

FIG. 9 depicts a plasmid map of pMON81465.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is the nucleic acid sequence of the granule bound starchsynthase from HOI001 (HOI001 GBSS from pMON72506).

SEQ ID NO: 2 is the published nucleic acid sequence of Zea mays GBSSfrom Shure et al., supra.

SEQ ID NO: 3 sets forth the predicted amino acid sequence of HOI001 GBSSfrom pMON72506.

SEQ ID NO: 4 sets forth the predicted amino acid sequence from the Zeamays GBSS as published by Shure et al., supra.

SEQ ID NO: 5 is a primer sequence for Primer number 14543.

SEQ ID NO: 6 is a primer sequence for Primer number 14547.

SEQ ID NO: 7 sets forth a nucleic acid sequence of a DNA molecule thatencodes a GBSS from corn line LH59.

SEQ ID NO: 8 sets forth the predicted amino acid sequence of GBSS fromcorn line LH59.

SEQ ID NO: 9 is a primer sequence for Primer number 20095.

SEQ ID NO: 10 is a primer sequence for Primer number 20092.

SEQ ID NO: 11 sets forth the coding region of the GBSS cDNA of HOI001.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are provided as an aid to understanding thedetailed description of the present invention.

The phrases “coding sequence,” “coding region,” “structural sequence,”and “structural nucleic acid sequence” refer to a physical structurecomprising an orderly arrangement of nucleotides. The nucleotides arearranged in a series of triplets that each form a codon. Each codonencodes a specific amino acid. Thus, the coding sequence, structuralsequence, and structural nucleic acid sequence encode a series of aminoacids forming a protein, polypeptide, or peptide sequence. The codingsequence, structural sequence, and structural nucleic acid sequence maybe contained within a larger nucleic acid molecule, vector, or the like.In addition, the orderly arrangement of nucleotides in these sequencesmay be depicted in the form of a sequence listing, figure, table,electronic medium, or the like.

The phrase “codon degeneracy” refers to divergence in the genetic codepermitting variation of the nucleotide sequence without affecting theamino acid sequence of an encoded polypeptide. Accordingly, the instantinvention relates to any nucleic acid fragment comprising a nucleotidesequence that encodes all or a substantial portion of the amino acidsequences set forth herein. The skilled artisan is well aware of the“codon-bias” exhibited by a specific host cell in usage of nucleotidecodons to specify a given amino acid. Therefore, when synthesizing anucleic acid fragment for improved expression in a host cell, it isdesirable to design the nucleic acid fragment such that its frequency ofcodon usage approaches the frequency of preferred codon usage of thehost cell.

The term “cDNA” refers to a double-stranded DNA that is complementary toand derived from mRNA.

The phrases “DNA sequence,” “nucleic acid sequence,” and “nucleic acidmolecule” refer to a physical structure comprising an orderlyarrangement of nucleotides. The DNA sequence or nucleotide sequence maybe contained within a larger nucleotide molecule, vector, or the like.In addition, the orderly arrangement of nucleic acids in these sequencesmay be depicted in the form of a sequence listing, figure, table,electronic medium, or the like.

“Expression” refers to the transcription of a gene to produce thecorresponding mRNA and translation of this mRNA to produce thecorresponding gene product (i.e., a peptide, polypeptide, or protein).

“Expression of antisense RNA” refers to the transcription of a DNA toproduce a first RNA molecule capable of hybridizing to a second RNAmolecule, which second RNA molecule encodes a gene product that isdesirably down-regulated.

As used herein, “gene” refers to a nucleic acid fragment that expressesa specific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. An “exogenous gene” or“transgene” refer to a non-native gene that has been introduced into thegenome by a transformation procedure.

“Hemizygous” refers to a diploid individual having only one copy of aparticular gene (for example, because a chromosome has been lost).“Homozygous” refers to a gene pair having identical alleles in twohomologous chromosomes.

“Heterologous” refers to the relationship between two or more nucleicacid or protein sequences that are derived from different sources. Forexample, a promoter is heterologous with respect to a coding sequence ifsuch a combination is not normally found in nature. In addition, aparticular sequence may be “heterologous” with respect to a cell ororganism into which it is inserted (i.e., does not naturally occur inthat particular cell or organism).

“Homology” refers to the level of similarity between two or more nucleicacid or amino acid sequences in terms of percent of positional identity(i.e., sequence similarity or identity). Homology also refers to theconcept of similar functional properties among different nucleic acidsor proteins.

“Hybridization” refers to the ability of a first strand of nucleic acidto join with a second strand via hydrogen bond base pairing when the twonucleic acid strands have sufficient sequence complementarity. As usedherein, a nucleic acid molecule is said to be the “complement” ofanother nucleic acid molecule if they exhibit complete complementarity.As used herein, molecules are said to exhibit “complete complementarity”when every nucleotide of one of the molecules is complementary to anucleotide of the other. Thus two nucleic acid strands are said to havesufficient complementarity when they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder appropriate conditions.

The phrases “marker-assisted selection” or “marker-assisted breeding”refer to the use of genetic markers to identify and select plants withsuperior phenotypic potential. Genetic markers determined previously tobe associated with a trait locus or trait loci are used to uncover thegenotype at trait loci by virtue of linkage between the marker locus andthe trait locus. Plants containing desired trait alleles are chosenbased upon their genotypes at linked marker loci.

The phrase “breeding population” refers to a genetically heterogeneouscollection of plants created for the purpose of identifying one or moreindividuals with desired phenotypic characteristics. The term“phenotype” refers to the observed expression of one or more plantcharacteristics.

A “genetic marker” is any morphological, biochemical, or nucleic acidbased phenotypic difference which reveals a DNA polymorphism. Examplesof genetic markers include but are not limited to RFLPs, RAPDs,allozymes, SSRs, and AFLPs.

The phrase “marker locus” refers to the genetically defined location ofDNA polymorphisms as revealed by a genetic marker. A “trait locus”refers to a genetically defined location for a collection of one or moregenes (alleles) which contribute to an observed characteristic.

The phrase “restriction fragment length polymorphism” (RFLP) refers to aDNA-based genetic marker in which size differences in restrictionendonuclease generated DNA fragments are observed via hybridization(Botstein et al., Am. J. Hum. Genet., 32:314-331 (1980)).

The phrase “random amplified polymorphic DNA” (RAPD) refers to a DNAamplification based genetic marker in which short, sequence arbitraryprimers are used and the resulting amplification products are sizeseparated and differences in amplification patterns observed (Williamset al., Nucleic Acids Res., 18:6531-6535 (1990)).

The phrase “simple sequence repeat” (SSR) refers to a DNAamplification-based genetic marker in which short stretches of tandemlyrepeated sequence motifs are amplified and the resulting amplificationproducts are size separated and differences in length of the nucleotiderepeat are observed (Tautz, Nucleic Acids Res., 112:4127-4138 (1989)).

The term “AFLP” refers to a DNA amplification-based genetic marker inwhich restriction endonuclease generated DNA fragments are ligated toshort DNA fragments which facilitate the amplification of the restrictedDNA fragments (Vos et al., Nucleic Acids Res., 23:4407-4414 (1995)). Theamplified fragments are size separated and differences in amplificationpatterns observed.

The phrase “operably linked” refers to the functional spatialarrangement of two or more nucleic acid regions or nucleic acidsequences. For example, a promoter region may be positioned relative toa nucleic acid sequence such that transcription of the nucleic acidsequence is directed by the promoter region. Thus, a promoter region is“operably linked” to the nucleic acid sequence.

The terms “promoter” or “promoter region” refer to a nucleic acidsequence, usually found upstream (5′) to a coding sequence that iscapable of directing transcription of a nucleic acid sequence into mRNA.The promoter or promoter region typically provides a recognition sitefor RNA polymerase and the other factors necessary for proper initiationof transcription. As contemplated herein, a promoter or promoter regionincludes variations of promoters derived by inserting or deletingregulatory regions, subjecting the promoter to random or site-directedmutagenesis, and the like. The activity or strength of a promoter may bemeasured in terms of the amounts of RNA it produces, or the amount ofprotein accumulation in a cell or tissue, relative to a second promoterthat is similarly measured.

The phrase “3′ non-coding sequences” refers to nucleotide sequenceslocated downstream of a coding sequence and include polyadenylationrecognition sequences and other sequences encoding regulatory signalscapable of affecting mRNA processing or gene expression. Thepolyadenylation signal is usually characterized by affecting theaddition of polyadenylic acid tracts to the 3′ end of the mRNAprecursor. The use of different 3′ non-coding sequences is exemplifiedby Ingelbrecht et al., Plant Cell, 1:671-680 (1989).

“Translation leader sequence” or “5′ untranslated region” or “5′-UTR”all refer to a nucleotide sequence located between the promoter sequenceof a gene and the coding sequence. The 5′-UTR is present in the fullyprocessed mRNA upstream of the translation start sequence. The 5′-UTRmay affect processing of the primary transcript to mRNA, mRNA stability,or translation efficiency. Examples of translation leader sequences havebeen described (Turner and Foster, Molecular Biotechnology,3:225(1995)).

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” (mRNA) refers tothe RNA that is without introns and that can be translated intopolypeptide by the cell. “Sense RNA” refers to an RNA transcript thatincludes the mRNA and so can be translated into a polypeptide by thecell. “Antisense RNA” refers to an RNA transcript that is complementaryto a target mRNA, resulting in specific RNA: RNA duplexes being formedby base pairing between the antisense RNA substrate and the target mRNA.

“Recombinant vector” refers to any agent by or in which a nucleic acidof interest is amplified, expressed, or stored, such as a plasmid,cosmid, virus, autonomously replicating sequence, phage, or linearsingle-stranded, circular single-stranded, linear double-stranded, orcircular double-stranded DNA or RNA nucleotide sequence. The recombinantvector may be derived from any source and is capable of genomicintegration or autonomous replication.

“Regulatory sequence” refers to a nucleotide sequence located upstream(5′), within, or downstream (3′) with respect to a coding sequence.Additionally, introns may have regulatory activity. Transcription andexpression of the coding sequence is typically impacted by the presenceor absence of the regulatory sequence.

“Substantially homologous” refers to two sequences that are at leastabout 90% identical in sequence, as measured by the CLUSTAL W method inthe Omiga program, using default parameters (Version 2.0; Accelrys, SanDiego, Calif.).

“Substantially purified” refers to a molecule separated fromsubstantially all other molecules normally associated with it in itsnative state. More preferably, a substantially purified molecule is thepredominant species present in a preparation. A substantially purifiedmolecule may be greater than about 60% free, preferably about 75% free,more preferably about 90% free, and most preferably about 95% free fromthe other molecules (exclusive of solvent) present in the naturalmixture. The phrase “substantially purified” is not intended toencompass molecules present in their native state.

The term “transformation” refers to the introduction of nucleic acidinto a recipient host. The term “host” refers to bacteria cells, fungi,animals or animal cells, plants or seeds, or any plant parts or tissuesincluding plant cells, protoplasts, calli, roots, tubers, seeds, stems,leaves, seedlings, embryos, and pollen.

As used herein, a “transgenic plant” is a plant having stably introducedinto its genome, for example, the nuclear or plastid genomes, a nucleicacid.

The terms “seeds” and “kernels” are understood to be equivalent inmeaning. The term kernel is frequently used in describing the seed of acorn or rice plant. In all plants the seed is the mature ovuleconsisting of a seed coat, embryo, and in plants of the presentinvention, an endosperm.

HOI001 GBSS Nucleic Acids

The present invention provides nucleic acids that encode polypeptidessubstantially homologous to a granule bound starch synthase isolatedfrom the inbred plant HOI001 (HOI001 GBSS). In one embodiment, thesenucleic acid molecules are used in the context of the present inventionfor increasing the oil content of plant tissues. In one embodiment, thepresent invention provides an isolated nucleic acid that encodes aHOI001 GBSS protein, which nucleic acid is selected from the groupconsisting of SEQ ID NO: 1 and complements thereof, and nucleic acidswhich encode polypeptides having at least about 94% sequence identitywith SEQ ID NO: 3. The percent sequence identity of the polypeptidesencoded by nucleic acids of this invention is preferably at least about95%; and most preferably at least about 98%.

The present invention also provides vectors containing such HOI001 GBSSnucleic acids. As set forth in further detail hereinbelow, preferrednucleic acids include appropriate regulatory elements operably linkedthereto that facilitate efficient expression of the inventive nucleicacids in a host, including without limitation bacterial, fungal, orplant hosts. Vectors useful in the context of the present invention caninclude such regulatory elements.

The nucleic acids and vectors encompassed by the present invention neednot have the exact nucleic acid sequences described herein. Instead, thesequences of these nucleic acids and vectors can vary, so long as thenucleic acid either performs the function for which it is intended orhas some other utility, for example, as a nucleic acid probe forcomplementary nucleic acids. For example, some sequence variability inany part of a HOI001 GBSS nucleic acid is permitted so long astransformation of a plant with the mutant or variant polypeptide orpolypeptides result in a phenotype substantially similar to that ofHOI001 GBSS. Most preferably, the aforementioned sequence variabilityresults in increased oil accumulation in plant tissues, as compared toplants of the same or similar genotype, but without the transgene.

Fragment and variant nucleic acids of SEQ ID NO: 1, are also encompassedby the present invention. Nucleic acid fragments encompassed by thepresent invention are of three general types. First, fragment nucleicacids that are not full length but do perform their intended functionare encompassed within the present invention. Second, fragments ofnucleic acids identified herein that are useful as hybridization probes,are also included in the invention. And, third, fragments of nucleicacids identified herein can be used in suppression technologies known inthe art, such as, for example, anti-sense technology or RNA inhibition(RNAi), which provides for reducing carbon flow in a plant into oil,making more carbon available for protein or starch accumulation, forexample. Thus, fragments of a nucleotide sequence, such as SEQ ID NO: 1may range from at least about 15 nucleotides, about 17 nucleotides,about 18 nucleotides, about 20 nucleotides, about 50 nucleotides, about100 nucleotides or more. In general, a fragment nucleic acid of thepresent invention can have any upper size limit so long as it is relatedin sequence to the nucleic acids of the present invention but does notinclude the full length.

As used herein, “variants” have substantially similar or substantiallyhomologous sequences when compared to reference or wild type sequence.For nucleotide sequences that encode proteins, variants also includethose sequences that, because of the degeneracy of the genetic code,encode the identical amino acid sequence of the reference protein.Variant nucleic acids also include those that encode polypeptides thatdo not have amino acid sequences identical to that of the proteinsidentified herein, but which encode an active protein with conservativechanges in the amino acid sequence.

The present invention is not limited to silent changes in the presentnucleotide sequences but also includes variant nucleic acid sequencesthat conservatively alter the amino acid sequence of a polypeptide ofthe present invention. Because it is the interactive capacity and natureof a protein that defines that protein's biological functional activity,certain amino acid sequence substitutions can be made in a proteinsequence and, of course, its underlying DNA coding sequence and,nevertheless, a protein with like properties can still be obtained. Itis thus contemplated by the inventors that various changes may be madein the peptide sequences of the proteins or fragments of the presentinvention, or corresponding DNA sequences that encode the peptides,without appreciable loss of their biological utility or activity.According to the present invention, then, variant and reference nucleicacids of the present invention may differ in the encoded amino acidsequence by one or more substitutions, additions, insertions, deletions,fusions, and truncations, which may be present in any combination, solong as an active HOI001 GBSS protein is encoded by the variant nucleicacid. Such variant nucleic acids will not encode exactly the same aminoacid sequence as the reference nucleic acid, but have conservativesequence changes. Codons capable of coding for such conservative aminoacid substitutions are well known in the art.

Another approach to identifying conservative amino acid substitutionsrequire analysis of the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biological function on a protein is generallyunderstood in the art (Kyte and Doolittle, J. Mol. Biol., 157:105-132(1982)). It is accepted that the relative hydropathic character of theamino acid contributes to the secondary structure of the resultantpolypeptide, which in turn defines the interaction of the protein withother molecules, for example, enzymes, substrates, receptors, DNA,antibodies, antigens, and the like.

Each amino acid has been assigned a hydropathic index on the basis ofits hydrophobicity and charge characteristics (Kyte and Doolittle, J.Mol. Biol., 157:105-132 (1982)); these are isoleucine (+4.5), valine(+4.2), leucine (+3.8), phenylalanine (+2.8), cysteine/cystine (+2.5),methionine (+1.9), alanine (+1.8), glycine (−0.4), threonine (−0.7),serine (−0.8), tryptophan (−0.9), tyrosine (−1.3), proline (−1.6),histidine (−3.2), glutamate (−3.5), glutamine (−3.5), aspartate (−3.5),asparagine (−3.5), lysine (−3.9), and arginine (−4.5).

In making such changes, the substitution of amino acids whosehydropathic indices are within ±2 is preferred, those that are within ±1are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, states that the greatest local average hydrophilicity ofa protein, as governed by the hydrophilicity of its adjacent aminoacids, correlates with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0),lysine (+3.0), aspartate (+3.0±1), glutamate (+3.0±1), serine (+0.3),asparagine (+0.2), glutamine (+0.2), glycine (0), threonine (−0.4),proline (−0.5±1), alanine (−0.5), histidine (−0.5), cysteine (−1.0),methionine (−1.3), valine (−1.5), leucine (−1.8), isoleucine (−1.8),tyrosine (−2.3), phenylalanine (−2.5), and tryptophan (−3.4).

In making such changes, the substitution of amino acids whosehydrophilicity values are within ±2 is preferred, those that are within±1 are particularly preferred, and those within ±0.5 are even moreparticularly preferred.

Variant nucleic acids with silent and conservative changes can bedefined and characterized by the degree of homology to the referencenucleic acid. Preferred variant nucleic acids are substantiallyhomologous to the reference nucleic acids of the present invention. Asrecognized by one of skill in the art, such substantially similarnucleic acids can hybridize under stringent conditions with thereference nucleic acids identified by SEQ ID NO: 1, herein. These typesof substantially homologous nucleic acids are encompassed by thisinvention.

Variant nucleic acids can be detected and isolated by standardhybridization procedures. Hybridization to detect or isolate suchsequences is generally carried out under “moderately stringent” andpreferably under “stringent” conditions. Moderately stringenthybridization conditions and associated moderately stringent andstringent hybridization wash conditions used in the context of nucleicacid hybridization experiments, such as Southern and northernhybridization, are sequence dependent, and are different under differentenvironmental parameters. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Laboratory Techniques in Biochemistry andMolecular Biology-Hybridization with Nucleic Acid Probes, page 1,Chapter 2 “Overview of principles of hybridization and the strategy ofnucleic acid probe assays” Elsevier, NY (1993). See also, J. Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press,NY, pp 9.31-9.58 (1989); J. Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, NY (3rd ed. 2001).

The present invention also provides methods for detection and isolationof derivative or variant nucleic acids encoding the proteins providedherein. The methods involve hybridizing at least a portion of a nucleicacid comprising any part of SEQ ID NO: 1 with respect to HOI001GBSS-related sequences, to a sample nucleic acid, thereby forming ahybridization complex; and detecting the hybridization complex. Thepresence of the complex correlates with the presence of a derivative orvariant nucleic acid that can be further characterized by nucleic acidsequencing, expression of RNA and/or protein and testing to determinewhether the derivative or variant retains the ability to increase oillevels in plant tissue when transformed into that plant. In general, theportion of a nucleic acid comprising any part of SEQ ID NO: 1 used forhybridization is preferably at least about fifteen nucleotides, andhybridization is under hybridization conditions that are sufficientlystringent to permit detection and isolation of substantially homologousnucleic acids; preferably, the hybridization conditions are “moderatelystringent”, more preferably the hybridization conditions are“stringent”, as defined herein and in the context of conventionalmolecular biological techniques well known in the art.

Generally, highly stringent hybridization and wash conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific double-stranded sequence at a defined ionic strengthand pH. For example, under “highly stringent conditions” or “highlystringent hybridization conditions” a nucleic acid will hybridize to itscomplement to a detectably greater degree than to other sequences (e.g.,at least 2-fold over background). By controlling the stringency of thehybridization and/or the washing conditions, nucleic acids having 100%complementary can be identified and isolated.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide, in which casehybridization temperatures can be decreased. Dextran sulfate and/orDenhardt's solution (50×Denhardt's is 5% Ficoll, 5%polyvinylpyrrolidone, 5% BSA) can also be included in the hybridizationreactions.

Exemplary low stringency conditions include hybridization with a buffersolution of 30 to 50% formamide, 5×SSC (20×SSC is 3M NaCl, 0.3 Mtrisodium citrate), 50 mM sodium phosphate, pH7, 5 mM EDTA, 0.1% SDS(sodium dodecyl sulfate), 5×Denhardt's with 100 μg/ml denatured salmonsperm DNA at 37° C., and a wash in 1× to 5×SSC (20×SSC defined as 3.0 MNaCl and 0.3 M trisodium citrate), 0.1% SDS at 37° C. Exemplary moderatestringency conditions include hybridization in 40 to 50% formamide,5×SSC 50 mM sodium phosphate, pH 7, 5 mM EDTA, 0.1% SDS, 5×Denhardt'swith 100 μg/ml denatured salmon sperm DNA at 42° C., and a wash in 0.1×to 2×SSC, 0.1% SDS at 42 to 55° C. Exemplary high stringency conditionsinclude hybridization in 50% formamide, 5×SSC, 50 mM sodium phosphate,pH 7.0, 5 mM EDTA, 0.1% SDS, 5×Denhardt's with 100 μg/ml denaturedsalmon sperm DNA at 42° C., and a wash in 0.1×SSC, 0.1% SDS at 60 to 65°C.

In another embodiment of the present invention, the inventive nucleicacids are defined by the percent identity relationship betweenparticular nucleic acids and other members of the class using analyticprotocols well known in the art. Such analytic protocols include, butare not limited to: CLUSTAL in the PC/Gene program (available fromIntelligenetics, Mountain View, Calif. or in the Omiga program version2.0 Accelrys Inc., San Diego, Calif.); the ALIGN program (Version 2.0);and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Version 8 (available from Genetics Computer Group(GCG), 575 Science Drive, Madison, Wis.). Alignments using theseprograms can be performed using the default parameters. The CLUSTALprogram is well described by Higgins et al., Gene, 73:237-244 (1988);Higgins et al., CABIOS, 5:151-153 (1989); Corpet et al., Nucleic AcidsRes., 16:10881-10890 (1988); Huang et al., CABIOS, 8:155-165 (1992); andPearson et al., Meth. Mol. Biol., 24:307-331 (1994). The ALIGN programis based on the algorithm of Meyers and Miller, Computer Applic. Biol.Sci., 4:11-17 (1988). The BLAST programs of Altschul et al., J. Mol.Biol., 215:403 (1990), are based on the algorithm of Karlin andAltschul, Proc. Natl. Acad. Sci. U.S.A., 87:2264-2268 (1990). To obtaingapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0)can be utilized as described in Altschul et al., Nucleic Acids Res.,25:3389 (1997). Alternatively, PSI-BLAST (in BLAST 2.0) can be used toperform an iterated search that detects distant relationships betweenmolecules. See, Altschul et al., supra. When utilizing BLAST, GappedBLAST, PSI-BLAST, the default parameters of the respective programs(e.g., BLASTN for nucleotide sequences, BLASTP for proteins) can beused. The BLASTN program (for nucleotide sequences) uses as defaults aword length (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5,N=−4, and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a word length (W) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix (see, Henikoff & Henikoff,Proc. Natl. Acad. Sci. U.S.A., 89:10915 (1989)) (see,http://www.ncbi.n1m.nih.gov.). Alignment may also be performed manuallyby inspection.

For purposes of the present invention, comparison of nucleotidesequences for determination of percent sequence identity to the nucleicacid sequences disclosed herein is preferably made using the BLASTNprogram (version 1.4.7 or later) with its default parameters or anyequivalent program. By “equivalent program” is intended any sequencecomparison program that, for any two sequences in question, generates analignment having identical nucleotide or amino acid residue matches andan identical percent sequence identity when compared to thecorresponding alignment generated by the preferred program.

Expression Vectors and Cassettes

The expression vectors and cassettes of the present invention includenucleic acids encoding HOI001 GBSS. A transgene comprising a HOI001 GBSScan be subcloned into an expression vector or cassette, and HOI001 GBSSexpression can be detected and/or quantified. This method of screeningis useful to identify transgenes providing for an expression of a HOI001GBSS, and expression of a HOI001 GBSS in a transformed plant cell.

Plasmid vectors that provide for easy selection, amplification, andtransformation of the transgene in prokaryotic and eukaryotic cellsinclude, for example, pUC-derived vectors, pSK-derived vectors,pGEM-derived vectors, pSP-derived vectors, pBS-derived vectors, pFastBac(Invitrogen Corporation, Carlsbad, Calif.) for baculovirus expressionand pYES2 (Invitrogen) for yeast expression. Additional elements may bepresent in such vectors, including origins of replication to provide forautonomous replication of the vector, selectable marker genes,preferably encoding antibiotic or herbicide resistance, unique multiplecloning sites providing for multiple sites to insert DNA sequences orgenes encoded in the transgene, and sequences that enhancetransformation of prokaryotic and eukaryotic cells. One vector that isuseful for expression in both plant and prokaryotic cells is the binaryTi plasmid (as disclosed in Schilperoot et al., U.S. Pat. No.4,940,838), as exemplified by vector pGA582. This binary Ti plasmidvector has been previously characterized by An, Methods in Enzymology,153:292 (1987). This binary Ti vector can be replicated in prokaryoticbacteria, such as E. coli and Agrobacterium. The Agrobacterium plasmidvectors can also be used to transfer the transgene to plant cells. Thebinary Ti vectors preferably include the T DNA right and left borders toprovide for efficient plant cell transformation, a selectable markergene, unique multiple cloning sites in the T border regions, the colE1replication of origin and a wide host range replicon. The binary Tivectors carrying a transgene of the present invention can be used totransform both prokaryotic and eukaryotic cells, but is preferably usedto transform plant cells, (see, Glassman et al., U.S. Pat. No.5,258,300). Examples of plant expression vectors include the commercialvectors pBI101, pBI101.2, pBI101.3, and pBIN19 (Clontech, Palo Alto,Calif.).

In general, the expression vectors and cassettes of the presentinvention contain at least a promoter capable of expressing RNA in aplant cell and a terminator, in addition to a nucleic acid encoding aHOI001 GBSS. Other elements may also be present in the expressioncassettes of the present invention. For example, expression cassettescan also contain enhancers, introns, untranslated leader sequences,cloning sites, matrix attachment regions for silencing the effects ofchromosomal control elements, and other elements known to one of skillin the art.

Expression cassettes have promoters that can regulate gene expression.Promoter regions are typically found in the flanking DNA sequenceupstream from coding regions in both prokaryotic and eukaryotic cells. Apromoter sequence provides for regulation of transcription of thedownstream gene sequence and typically includes from about 50 to about2,000 nucleotide base pairs. Promoter sequences also contain regulatorysequences, such as enhancer sequences that can influence the level ofgene expression. Some isolated promoter sequences can provide for geneexpression of heterologous genes, that is, a gene different from thenative or homologous gene. Promoter sequences are also known to bestrong or weak or inducible. A strong promoter provides for a high levelof gene expression, whereas a weak promoter provides for a very lowlevel of gene expression. An inducible promoter is a promoter thatprovides for turning on and off of gene expression in response to anexogenously added agent or to an environmental or developmentalstimulus. Promoters can also provide for tissue specific ordevelopmental regulation. An isolated promoter sequence that is a strongpromoter for heterologous genes is advantageous because it provides fora sufficient level of gene expression to allow for easy detection andselection of transformed cells and provides for a high level of geneexpression when desired. Transcription initiation regions that arepreferentially expressed in seed tissue, and that are undetectable inother plant parts, are considered desirable for seed oil modificationsin order to minimize any disruptive or adverse effects of the geneproduct.

Promoters of the present invention will generally include, but are notlimited to, promoters that function in bacteria, plant cells, orplastids. Useful promoters for bacterial expression are the lacZ, T7,T5, or E. coli glg C promoters. Useful promoters for plant cells includewheat high molecular weight glutenin promoter (bp 2647-3895 of GenbankAccession X12928, version X12928.3, originally described in Anderson etal., Nucleic Acids Res., 17:461-462 (1989)), the globulin promoter (see,Belanger and Kriz, Genet., 129:863-872, (1991)), gamma zein Z27 promoter(see, U.S. Ser. No. 08/763,705; also, Lopes et al., Mol Gen Genet.,247:603-613 (1995)), L3 oleosin promoter (U.S. Pat. No. 6,433,252), CaMV35S promoter (Odell et al., Nature, 313:810 (1985)), the CaMV 19S(Lawton et al., Plant Mol. Biol., 9:31F (1987)), nos (Ebert et al.,Proc. Natl. Acad. Sci. U.S.A., 84:5745 (1987)), Adh (Walker et al.,Proc. Natl. Acad. Sci. U.S.A., 84:6624 (1987)), sucrose synthase (Yanget al., Proc. Natl. Acad. Sci. U.S.A., 87:4144 (1990)), tubulin, actin(Wang et al., Mol. Cell. Biol., 12:3399 (1992)), cab (Sullivan et al.,Mol. Gen. Genet., 215:431 (1989)), PEPCase promoter (Hudspeth et al.,Plant Mol. Biol., 12:579 (1989)), or those associated with the R genecomplex (Chandler et al., The Plant Cell, 1:1175 (1989)).

Indeed in a preferred embodiment the promoter used is highly-expressedin the endosperm. Exemplary promoters include those from the zeins whichare a group of storage proteins found in maize endosperm. Genomic clonesfor zein genes have been isolated (Pedersen et al., Cell, 29:1015-1026(1982) and Russell et al., Transgenic Res., 6(2):157-168 (1997)) and thepromoters from these clones, including the 15 kD, 16 kD, 19 kD, 22 kD,and 27 kD genes (Z27, U.S. Ser. No. 08/763,705; also, Reina et al.,Nucl. Acids Res., 18:6426 (1990), Lopes et al., Mol. Gen. Genet.,247:603-613 (1995)), can also be used. Other preferred promoters, knownto function in maize, and in other plants, include the promoters for thefollowing genes: WAXY (granule bound starch synthase; Shure et al.,Cell, 35:225-233 (1983); Russell et al., Transgenic Res., 6(2):157-168(1997)), Brittle 2 and Shrunken 2 (ADP glucose pryophosphorylase,Anderson et al., Gene, 97:199-205 (1991), Russell et al., TransgenicRes., 6(2):157-168 (1997)), Shrunken 1(sucrose synthase, Yang andRussell, Proc. Natl. Acad. Sci. U.S.A., 87:4144-4148 (1990)), branchingenzymes I and II, WAXY promoter from rice (Terada et al., Plant CellPhysiology, 41(7):881-888 (2000)), debranching enzymes, glutelins (Zhenget al., Plant J., 4:357-366 (1993), Russell et al., Transgenic Res.,6(2):157-168 (1997)), and Betl1 (basal endosperm transfer layer; Hueroset al., Plant Physiol., 121:1143-1152 (1999)). Other promoters useful inthe practice of the present invention that are known by one of skill inthe art are also contemplated by the invention.

Moreover, transcription enhancers or duplications of enhancers can beused to increase expression from a particular promoter. Examples of suchenhancers include, but are not limited to, elements from the CaMV 35Spromoter and octopine synthase genes (Last et al., U.S. Pat. No.5,290,924). As the DNA sequence between the transcription initiationsite and the start of the coding sequence, i.e., the untranslated leadersequence, can influence gene expression, one may also wish to employ aparticular leader sequence. Any leader sequence available to one ofskill in the art may be employed. Preferred leader sequences directoptimum levels of expression of the attached gene, for example, byincreasing or maintaining mRNA stability and/or by preventinginappropriate initiation of translation (Joshi, Nucl. Acid Res., 15:6643(1987)). The choice of such sequences is at the discretion of those ofskill in the art. Sequences that are derived from genes that are highlyexpressed in higher plants, and in soybean, corn, and canola inparticular, are contemplated.

Expression cassettes of the present invention will also include asequence near the 3′ end of the cassette that acts as a signal toterminate transcription from a heterologous nucleic acid and thatdirects polyadenylation of the resultant mRNA. These are commonlyreferred to as 3′ untranslated regions or 3′ UTRs. Some 3′ elements thatcan act as transcription termination signals include the wheat HSP17 3′UTR (bp532-741 of GenBank X13431, version X13431.1, McElvain and Spiker,Nucleic Acids Res., 17:1764 (1989)), those from the nopaline synthasegene of Agrobacterium tumefaciens (Bevan et al., Nucl. Acid Res., 11:369(1983)), a napin 3′ UTR (Kridl et al., Seed Sci Res., 1:209-219 (1991)),a globulin 3′ UTR (Belanger and Kriz, Genetics, 129:863-872 (1991)), orone from a zein gene, such as Z27 (Lopes et al., Mol Gen Genet.,247:603-613 (1995)). Other 3′ elements known by one of skill in the artalso can be used in the vectors of the present invention.

Regulatory elements, such as Adh intron 1 (Callis et al., GenesDevelop., 1:1183 (1987)), a rice actin intron (McElroy et al., Mol. Gen.Genet., 231(1):150-160 (1991)), sucrose synthase intron (Vasil et al.,Plant Physiol., 91:5175 (1989)), the maize HSP70 intron (Rochester etal., EMBO J., 5:451-458 (1986)), or TMV omega element (Gallie et al.,The Plant Cell, 1:301 (1989)) may further be included where desired.These 3′ nontranslated regulatory sequences can be obtained as describedin An, Methods in Enzymology, 153:292 (1987) or are already present inplasmids available from commercial sources, such as Clontech, Palo Alto,Calif. The 3′ nontranslated regulatory sequences can be operably linkedto the 3′ terminus of any heterologous nucleic acid to be expressed bythe expression cassettes contained within the present vectors. Othersuch regulatory elements useful in the practice of the present inventionare known by one of skill in the art and can also be placed in thevectors of the invention.

The vectors of the present invention, as well as the coding regionsclaimed herein, can be optimized for expression in plants by having oneor more codons replaced by other codons encoding the same amino acids sothat the polypeptide is optimally translated by the translationmachinery of the plant species in which the vector is used.

Selectable Markers

Selectable marker genes or reporter genes are also useful in the presentinvention. Such genes can impart a distinct phenotype to cellsexpressing the marker gene and thus allow such transformed cells to bedistinguished from cells that do not have the marker. Selectable markergenes confer a trait that one can “select” for by chemical means, i.e.,through the use of a selective agent (e.g., a herbicide, antibiotic, orthe like). Reporter genes, or screenable genes, confer a trait that onecan identify through observation or testing, i.e., by “screening” (e.g.,the R-locus trait). Of course, many examples of suitable marker genesare known to the art and can be employed in the practice of the presentinvention.

A number of selectable marker genes are known in the art and can be usedin the present invention. A preferred selectable marker gene for use inthe present invention would include genes that confer resistance toherbicides like glyphosate, such as EPSP (Della-Cioppa et al.,Bio/Technology, 5(6):579-84 (1987)). A particularly preferred selectablemarker would include a gene that encodes an altered EPSP synthaseprotein (Hinchee et al., Biotech., 6:915 (1988)). Other possibleselectable markers for use in connection with the present inventioninclude, but are not limited to, a neo gene (Potrykus et al., Mol. Gen.Genet., 199:183 (1985)) which codes for kanamycin resistance and can beselected for by applying kanamycin, a kanamycin analog such as geneticin(Sigma Chemical Company, St. Louis, Mo.), and the like; a bar gene thatcodes for bialaphos resistance; a nitrilase gene, such as bxn fromKlebsiella ozaenae, which confers resistance to bromoxynil (Stalker etal., Science, 242:419 (1988)); a mutant acetolactate synthase gene (ALS)that confers resistance to imidazolinone, sulfonylurea or otherALS-inhibiting chemicals (EP 154 204A1 (1985)); a methotrexate-resistantDHFR gene (Thillet et al., J. Biol. Chem., 263:12500 (1988)); a dalapondehalogenase gene that confers resistance to the herbicide dalapon.Where a mutant EPSP synthase gene is employed, additional benefit may berealized through the incorporation of a suitable plastid transit peptide(CTP).

Screenable markers that may be employed include, but are not limited to,β-glucuronidase or uidA gene (GUS), which encodes an enzyme for whichvarious chromogenic substrates are known; an R-locus gene, which encodesa product that regulates the production of anthocyanin pigments (redcolor) in plant tissues (Dellaporta et al., In Chromosome Structure andFunction, pp. 263-282 (1988)); a β-lactamase gene (Sutcliffe, Proc.Natl. Acad. Sci. U.S.A., 75:3737 (1978)), which encodes an enzyme forwhich various chromogenic substrates are known (e.g., PADAC, achromogenic cephalosporin); a xylE gene (Zukowsky et al., Proc. Natl.Acad. Sci. U.S.A., 80:1101 (1983)) that encodes a catechol dioxygenasethat can convert chromogenic catechols; an α-amylase gene (Ikuta et al.,Biotech., 8:241 (1990)); a tyrosinase gene (Katz et al., J. Gen.Microbiol., 129:2703 (1983)) that encodes an enzyme capable of oxidizingtyrosine to DOPA and dopaquinone, which in turn condenses to form theeasily detectable compound melanin; a β-galactosidase gene, whichencodes an enzyme for which there are chromogenic substrates; aluciferase (lux) gene (Ow et al., Science, 234:856 (1986)), which allowsfor bioluminescence detection; or an aequorin gene (Prasher et al.,Biochem. Biophys. Res. Comm., 126:1259 (1985)), which may be employed incalcium-sensitive bioluminescence detection, or a green fluorescentprotein gene (Niedz et al., Plant Cell Reports, 14:403 (1995)). In apreferred embodiment, the screenable marker gene is operably linked toan aleurone-specific promoter as described by Kriz et al., in U.S. Pat.No. 6,307,123.

In addition to nuclear plant transformation, the present invention alsoextends to direct transformation of the plastid genome of plants. Hence,targeting of the gene product to an intracellular compartment withinplant cells may also be achieved by direct delivery of a gene to theintracellular compartment. In some embodiments, direct transformation ofplastid genome may provide additional benefits over nucleartransformation. For example, direct plastid transformation of HOI001GBSS eliminates the requirement for a plastid targeting peptide andpost-translational transport and processing of the pre-protein derivedfrom the corresponding nuclear transformants. Plastid transformation ofplants has been described by P. Maliga, Current Opinion in PlantBiology, 5:164-172 (2002), Heifetz, Biochimie, 82:655-666 (2000), Bock,J. Mol. Biol., 312:425-438 (2001), and Daniell et al., Trends in PlantScience, 7:84-91 (2002), and references cited therein.

After constructing a transgene containing an HOI001 GBSS, the expressionvector or cassette can then be introduced into a plant cell. Dependingon the type of plant cell, the level of gene expression, and theactivity of the enzyme encoded by the gene, introduction of DNA encodingan HOI001 GBSS into the plant cell can lead to increased oil content inplant tissues.

Plant Transformation

Techniques for transforming a plant cell, a plant tissue, a plant organ,or a plant with a nucleic acid construct, such as a vector are known inthe art. Such methods involve plant tissue culture techniques, forexample. As used herein, “transforming” refers to the introduction ofnucleic acid into a recipient host and the expression therein.

The plant cell, plant tissue, plant organ, or plant can be contactedwith the vector by any suitable means as known in the art. Preferably, atransgenic plant expressing the desired protein is to be produced.Various methods for the introduction of a desired polynucleotidesequence encoding the desired protein into plant cells include, but arenot limited to: (1) physical methods such as microinjection (Capecchi,Cell, 22(2):479-488 (1980)), electroporation (Fromm et al., Proc. Nat.Acad. Sci. U.S.A., 82(17):5824-5828 (1985); U.S. Pat. No. 5,384,253),and microprojectile bombardment mediated delivery (Christou et al.,Bio/Technology, 9:957 (1991); Fynan et al., Proc. Nat. Acad. Sci.U.S.A., 90(24):11478-11482 (1993)); (2) virus mediated delivery methods(Clapp, Clin. Perinatol., 20(1):155-168 (1993); Lu et al., J. Exp. Med.,178(6):2089-2096 (1993); Eglitis and Anderson, Biotechniques,6(7):608-614 (1988); and (3) Agrobacterium-mediated transformationmethods.

The most commonly used methods for transformation of plant cells are theAgrobacterium-mediated DNA transfer process (Fraley et al., Proc. Nat.Acad. Sci. U.S.A., 80:4803 (1983)) and the microprojectile bombardmentmediated process. Typically, nuclear transformation is desired but whereit is desirable to specifically transform plastids, such as chloroplastsor amyloplasts, plant plastids may be transformed utilizing amicroprojectile bombardment mediated delivery of the desiredpolynucleotide for certain plant species such as tobacco, Arabidopsis,potato, and Brassica species.

Agrobacterium-mediated transformation is achieved through the use of agenetically engineered soil bacterium belonging to the genusAgrobacterium. Several Agrobacterium species mediate the transfer of aspecific DNA known as “T-DNA,” which can be genetically engineered tocarry any desired piece of DNA into many plant species. The major eventsmarking the process of T-DNA mediated pathogenesis are: induction ofvirulence genes, processing, and transfer of T-DNA. This process is thesubject of many reviews (Ream, Ann. Rev. Phytopathol., 27:583-618(1989); Howard and Citovsky, Bioassays, 12:103-108 (1990); Kado, Crit.Rev. Plant Sci., 10:1-32 (1991); Zambryski, Annual Rev. Plant Physiol.Plant Mol. Biol., 43:465-490 (1992); Gelvin, In Transgenic Plants, Kungand Wu, (eds.), Academic Press, San Diego, Calif., pp. 49-87 (1993);Binns and Howitz, In Bacterial Pathogenesis of Plants and Animals, Dang,(ed.). Berlin: Springer Verlag, pp. 119-138 (1994); Hooykaas andBeijersbergen, Ann. Rev. Phytopathol., 32:157-179 (1994); Lessl andLanka, Cell, 77:321-324 (1994); Zupan and Zambryski, Annual Rev.Phytopathol., 27:583-618 (1995)).

Agrobacterium-mediated genetic transformation of plants involves severalsteps. The first step, in which the virulent Agrobacterium and plantcells are first brought into contact with each other, is generallycalled “inoculation.” The Agrobacterium containing solution is thenremoved from contact with the explant by draining or aspiration.Following the inoculation, the Agrobacterium and plant cells/tissues arepermitted to be grown together for a period of several hours to severaldays or more under conditions suitable for growth and T-DNA transfer.This step is termed “co-culture.” Following co-culture and T-DNAdelivery, the plant cells are treated with bactericidal orbacteriostatic agents to kill the Agrobacterium remaining in contactwith the explant and/or in the vessel containing the explant. If this isdone in the absence of any selective agents to promote preferentialgrowth of transgenic versus non-transgenic plant cells, then this istypically referred to as the “delay” step. If done in the presence ofselective pressure favoring transgenic plant cells, then it is referredto as a “selection” step. When a “delay” is used, it is typicallyfollowed by one or more “selection” steps. Both the “delay” and“selection” steps typically include bactericidal or bacteriostaticagents to kill any remaining Agrobacterium cells because the growth ofAgrobacterium cells is undesirable after the infection (inoculation andco-culture) process.

A number of wild-type and disarmed strains of Agrobacterium tumefaciensand Agrobacterium rhizogenes harboring Ti or Ri plasmids can be used forgene transfer into plants. The Agrobacterium hosts contain disarmed Tiand Ri plasmids that do not contain the oncogenes that causetumorigenesis or rhizogenesis, respectfully, which are used as thevectors and contain the genes of interest that are subsequentlyintroduced into plants. Preferred strains would include but are notlimited to Agrobacterium tumefaciens strain C58, a nopaline-type strainthat is used to mediate the transfer of DNA into a plant cell,octopine-type strains such as LBA4404 or succinamopine-type strains,e.g., EHA101 or EHA105. The nucleic acid molecule, prepared as a DNAcomposition in vitro, is introduced into a suitable host such as E. coliand mated into the Agrobacterium, or directly transformed into competentAgrobacterium. These techniques are well-known to those of skill in theart.

The Agrobacterium can be prepared either by inoculating a liquid such asLuria Burtani (LB) media directly from a glycerol stock or streaking theAgrobacterium onto a solidified media from a glycerol stock, allowingthe bacteria to grow under the appropriate selective conditions,generally from about 26° C.-30° C., or about 28° C., and taking a singlecolony or a small loop of Agrobacterium from the plate and inoculating aliquid culture medium containing the selective agents. Those of skill inthe art are familiar with procedures for growth and suitable cultureconditions for Agrobacterium as well as subsequent inoculationprocedures. The density of the Agrobacterium culture used forinoculation and the ratio of Agrobacterium cells to explant can varyfrom one system to the next, and therefore optimization of theseparameters for any transformation method is expected.

Typically, an Agrobacterium culture is inoculated from a streaked plateor glycerol stock and is grown overnight and the bacterial cells arewashed and resuspended in a culture medium suitable for inoculation ofthe explant.

With respect to microprojectile bombardment (U.S. Pat. Nos. 5,550,318;5,538,880; and 5,610,042; and PCT Publication WO 95/06128; each of whichis specifically incorporated herein by reference in its entirety),particles are coated with nucleic acids and delivered into cells by apropelling force. Exemplary particles include those comprised oftungsten, platinum, and preferably, gold. It is contemplated that insome instances DNA precipitation onto metal particles would not benecessary for DNA delivery to a recipient cell using microprojectilebombardment. However, it is contemplated that particles may contain DNArather than be coated with DNA. Hence, it is proposed that DNA-coatedparticles may increase the level of DNA delivery via particlebombardment but are not, in and of themselves, necessary.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos or other targetcells may be arranged on solid culture medium. The cells to be bombardedare positioned at an appropriate distance below the microprojectilestopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by microprojectile bombardment is the Biolistics Particle DeliverySystem (BioRad, Hercules, Calif.), which can be used to propel particlescoated with DNA or cells through a screen, such as a stainless steel orNytex screen, onto a filter surface covered with monocot plant cellscultured in suspension. The screen disperses the particles so that theyare not delivered to the recipient cells in large aggregates. It isbelieved that a screen intervening between the projectile apparatus andthe cells to be bombarded reduces the size of projectile aggregates andmay contribute to a higher frequency of transformation by reducing thedamage inflicted on the recipient cells by projectiles that are toolarge.

For microprojectile bombardment, one will attach (i.e., “coat”) DNA tothe microprojectiles such that it is delivered to recipient cells in aform suitable for transformation thereof. In this respect, at least someof the transforming DNA must be available to the target cell fortransformation to occur, while at the same time during delivery the DNAmust be attached to the microprojectile. Therefore, availability of thetransforming DNA from the microprojectile may comprise the physicalreversal of bonds between transforming DNA and the microprojectilefollowing delivery of the microprojectile to the target cell. This neednot be the case, however, as availability to a target cell may occur asa result of breakage of unbound segments of DNA or of other moleculeswhich comprise the physical attachment to the microprojectile.Availability may further occur as a result of breakage of bonds betweenthe transforming DNA and other molecules, which are either directly orindirectly attached to the microprojectile. It further is contemplatedthat transformation of a target cell may occur by way of directrecombination between the transforming DNA and the genomic DNA of therecipient cell. Therefore, as used herein, a “coated” microprojectilewill be one which is capable of being used to transform a target cell,in that the transforming DNA will be delivered to the target cell, yetwill be accessible to the target cell such that transformation mayoccur.

Any technique for coating microprojectiles, which allows for delivery oftransforming DNA to the target cells, may be used. Methods for coatingmicroprojectiles, which have been demonstrated to work well with thepresent invention, have been specifically disclosed herein. DNA may bebound to microprojectile particles using alternative techniques,however. For example, particles may be coated with streptavidin and DNAend labeled with long chain thiol cleavable biotinylated nucleotidechains. The DNA adheres to the particles due to the streptavidin-biotininteraction, but is released in the cell by reduction of the thiollinkage through reducing agents present in the cell.

Alternatively, particles may be prepared by functionalizing the surfaceof a gold oxide particle, providing free amine groups. DNA, having astrong negative charge, binds to the functionalized particles.Furthermore, charged particles may be deposited in controlled arrays onthe surface of mylar flyer disks used in the PDS-1000 Biolistics device,thereby facilitating controlled distribution of particles delivered totarget tissue.

As disclosed above, it further is proposed, that the concentration ofDNA used to coat microprojectiles may influence the recovery oftransformants containing a single copy of the transgene. For example, alower concentration of DNA may not necessarily change the efficiency ofthe transformation, but may instead increase the proportion of singlecopy insertion events. In this regard, approximately 1 ng to 2000 ng oftransforming DNA may be used per each 1.8 mg of startingmicroprojectiles. In other embodiments of the present invention,approximately 2.5 ng to 1000 ng, 2.5 ng to 750 ng, 2.5 ng to 500 ng, 2.5ng to 250 ng, 2.5 ng to 100 ng, or 2.5 ng to 50 ng of transforming DNAmay be used per each 1.8 mg of starting microprojectiles.

Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species. Examples of species thathave been transformed by microprojectile bombardment include monocotspecies such as maize (PCT Publication WO 95/06128), barley, wheat (U.S.Pat. No. 5,563,055, specifically incorporated herein by reference in itsentirety), rice, oat, rye, sugarcane, and sorghum; as well as a numberof dicots including tobacco, soybean (U.S. Pat. No. 5,322,783,specifically incorporated herein by reference in its entirety),sunflower, peanut, cotton, tomato, and legumes in general (U.S. Pat. No.5,563,055, specifically incorporated herein by reference in itsentirety).

For microprojectile bombardment transformation in accordance with thepresent invention, both physical and biological parameters may beoptimized. Physical factors are those that involve manipulating theDNA/microprojectile precipitate or those that affect the flight andvelocity of either the macro- or microprojectiles. Biological factorsinclude all steps involved in manipulation of cells before andimmediately after bombardment, such as the osmotic adjustment of targetcells to help alleviate the trauma associated with bombardment, theorientation of an immature embryo or other target tissue relative to theparticle trajectory, and also the nature of the transforming DNA, suchas linearized DNA or intact supercoiled plasmids. It is believed thatpre-bombardment manipulations are especially important for successfultransformation of immature embryos.

Accordingly, it is contemplated that one may wish to adjust variousbombardment parameters in small scale studies to fully optimize theconditions. One may particularly wish to adjust physical parameters suchas DNA concentration, gap distance, flight distance, tissue distance,and helium pressure. It further is contemplated that the grade of heliummay affect transformation efficiency. One also may optimize the traumareduction factors (TRFs) by modifying conditions which influence thephysiological state of the recipient cells and which may thereforeinfluence transformation and integration efficiencies. For example, theosmotic state, tissue hydration, and the subculture stage or cell cycleof the recipient cells may be adjusted for optimum transformation.

Other methods of cell transformation can also be used and include butare not limited to introduction of DNA into plants by direct DNAtransfer into pollen (Hess et al., Intern Rev. Cytol., 107:367 (1987);Luo et al., Plant Mol. Biol. Reporter, 6:165 (1988)), by directinjection of DNA into reproductive organs of a plant (Pena et al.,Nature, 325:274 (1987)), or by direct injection of DNA into the cells ofimmature embryos followed by the rehydration of desiccated embryos(Neuhaus et al., Theor. Appl. Genet., 75:30 (1987)).

The regeneration, development, and cultivation of plants from singleplant protoplast transformants or from various transformed explants iswell known in the art (Weissbach and Weissbach, In: Methods for PlantMolecular Biology, Academic Press, San Diego, Calif., (1988)). Thisregeneration and growth process typically includes the steps ofselection of transformed cells, culturing those individualized cellsthrough the usual stages of embryonic development through the rootedplantlet stage. The resulting transgenic rooted shoots are thereafterplanted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the exogenous genethat encodes a protein of interest is well known in the art. Preferably,the regenerated plants are self-pollinated to provide homozygoustransgenic plants. Otherwise, pollen obtained from the regeneratedplants is crossed to seed-grown plants of agronomically important lines.Conversely, pollen from plants of these important lines is used topollinate regenerated plants. A transgenic plant of the presentinvention containing a desired polypeptide is cultivated using methodswell known to one skilled in the art.

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated.

Assays for gene expression based on the transient expression of clonednucleic acid constructs have been developed by introducing the nucleicacid molecules into plant cells by polyethylene glycol treatment,electroporation, or particle bombardment (Marcotte et al., Nature,335:454-457 (1988); Marcotte et al., Plant Cell, 1:523-532 (1989);McCarty et al., Cell, 66:895-905 (1991); Hattori et al., Genes Dev.,6:609-618 (1992); Goff et al., EMBO J., 9:2517-2522 (1990)). Transientexpression systems may be used to functionally dissect gene constructs(see generally, Maliga et al., Methods in Plant Molecular Biology, ColdSpring Harbor Press (1995)).

Any of the nucleic acid molecules of the present invention may beintroduced into a plant cell in a permanent or transient manner incombination with other genetic elements such as vectors, promoters,enhancers, etc. Further, any of the nucleic acid molecules of thepresent invention may be introduced into a plant cell in a manner thatallows for expression or overexpression of the protein or fragmentthereof encoded by the nucleic acid molecule.

Transgenic plants may find use in the commercial manufacture of proteinsor other molecules, such as oils, where the molecules of interest areextracted or purified from plant parts, seeds, and the like. Cells ortissue from the plants may also be cultured, grown in vitro, orfermented to manufacture such molecules.

Improvements encoded by the recombinant DNA may be transferred, e.g.,from cells of one species to cells of other species, e.g., by protoplastfusion. The transgenic plants may also be used in commercial breedingprograms, or may be crossed or bred to plants of related crop species.For example, a nucleic acid of the present invention, operably linked toa promoter, can be introduced into a particular plant variety bycrossing, without the need for ever directly transforming a plant ofthat given variety. Therefore the present invention not only encompassesa plant directly regenerated from cells that have been transformed inaccordance with the present invention, but also the progeny of suchplants.

The present invention also provides for a method of stably expressing anHOI001 GBSS of interest in a plant, which includes, contacting the plantcell with a vector of the present invention that has a nucleic acidencoding the HOI001 GBSS of interest, under conditions effective totransfer and integrate the vector into the nuclear genome of the cell. Apromoter within the expression cassette can be any of the promotersprovided herein, for example, a constitutive promoter, an induciblepromoter, a tissue-specific promoter, or a seed specific promoter. Suchpromoters can provide expression of an encoded HOI001 GBSS at a desiredtime, or at a desired developmental stage, or in a desired tissue. Thevector can also include a selectable marker gene. When using the vectorwith Agrobacterium tumefaciens, the vector can have an Agrobacteriumtumefaciens origin of replication.

Plants

Plants for use with the vectors of the present invention preferablyinclude monocots, especially oil producing species, most preferably corn(Zea mays). Other species contemplated by the present invention includealfalfa (Medicago sativa), rice (Oryza sativa), barley (Hordeumvulgare), millet (Panicum miliaceum), rye (Secale cereale), wheat(Triticum aestivum), and sorghum (Sorghum bicolor).

Any of the plants or parts thereof of the present invention may beprocessed to produce a feed, meal, protein, or oil preparation. Aparticularly preferred plant part for this purpose is a seed. Methods toproduce feed, meal, protein, and oil preparations are known in the art.See, for example, U.S. Pat. Nos. 4,957,748; 5,100,679; 5,219,596;5,936,069; 6,005,076; 6,146,669; and 6,156,227.

Characterization of Transformed Plants

To confirm the presence of the transgene in the regenerated plant, avariety of techniques, which are well known in the art, are available.Examples of these techniques include but are not limited to: (a)molecular assays of DNA integration or RNA expression such as Southernor northern blotting, TAQMAN® technology (Applied Biosystems, FosterCity, Calif.) and PCR; (b) biochemical assays detecting the presence ofthe protein product such as ELISA, western blotting, or by enzymaticfunction; or (c) chemical analysis of the targeted plant part, such asseed tissue, for qualitative and quantitative determination of oil,protein, or starch.

The following examples are provided to illustrate the present inventionand are not intended to limit the invention in any way.

EXAMPLE 1

This example describes the isolation and sequencing of the HOI001 GBSSgene from corn line HOI001. HOI001 is an inbred plant derived from MGSC915E (Maize Genetic Stock Center, Urbana, Ill.), and is more fullydescribed in U.S. Patent Publication Nos. 20030172416 and 20030154524,both of which are incorporated herein by reference.

Genomic DNA was extracted from corn germ tissue from HOI001, 22 daysafter pollination, using the following procedure. Between 50-100 mgdissected germ tissue was placed in a Bio101 Multimix tube (Qiagen,Carlsbad, Calif., Cat. No. 657-601) with extraction buffer and glassbeads. The extraction buffer consisted of 100 mM Tris-HCl (pH 8.0), 50mM EDTA, 100 mM NaCl, 5 mM DTT, and 1% SDS. The tissue was thendisrupted using the Bio 101 FASTPREP® machine (Qiagen) with 3 pulses of20 seconds each. Following a 15 minute incubation at 65° C., 330 μl of5M potassium acetate was added to each tube. The tubes were thenincubated at 0° C. for 20 minutes to precipitate the SDS, followed bycentrifugation at 12,000 rpm (Eppendorf Model 54172) for 10 minutes. Thesupernatant was transferred to a new tube and 100 μl of 5M ammoniumacetate (pH 7.0) and 700 μl of isopropanol was added to precipitate theDNA. The tubes were mixed by inversion and centrifuged at 14,000 rpm for10 minutes. After discarding the supernatant, the pellet was resuspendedin 500 μl of 70% ethanol and recovered by centrifugation at 14,000 rpmfor 5 minutes. The recovered pellet containing the DNA was resuspendedin 50 μl of TE buffer and stored at 4° C.

The HOI001 GBSS gene was isolated from the extracted genomic DNA usingPCR methodology that was adapted from Advantage GC (BD BiosciencesClontech, Palo Alto, Calif.). The following primers were designed basedon the published sequence of Zea mays GBSS from Shure et al., Cell,35(1):225-233 (1983) [SEQ ID NO: 2]: 5′ primer (Primer number 14543)[SEQ ID NO: 5] 5′-TCAGCCGTTCGTGTGGCAAGATTCATCTGTTGTCTC-3′ 3′ primer(Primer number 14547) [SEQ ID NO: 6]5′-TCAGCGGGATTATTTACTCCACCACTACAGGTCCATTT-3′.

The following PCR reaction was assembled for a total volume of 50 μl; 37 μl PCR grade water   5 μl 5X Advantage GC PCR buffer   1 μl 50X dNTPMix (10 mM each)   1 μl 50X Advantage GC Polymerase Mix 2.5 μl primer14543 2.5 μl primer 14547   1 μl genomic DNA

The cycle parameters were: 95° C. for 1 minute, 35 cycles of 95° C. for30 seconds and 68° C. for 3 minutes.

The PCR products were separated by electrophoresis in agarose and a 4.7kB fragment containing the gene of interest was observed. Fivemicroliters of the original PCR reaction was utilized as template foradditional amplification using the same primers and conditions describedabove. The 4.7 kB amplification products from independent amplificationreactions were isolated by agarose gel electrophoresis, cloned into thePCR 2.1 cloning vector using the TOPO TA cloning kit (Invitrogen), thentransformed into an E. coli host. Plasmid DNA was prepared from culturesgrown from each colony, and then the inserts from 3 separate plasmidpreparations were sequenced. Alignment of these sequences generated aconsensus sequence highly homologous but not identical to the publishedGBSS gene, although no specific insert sequence was equivalent to theconsensus. One clone (designated pCGN9480-2) had an insert sequence withthe fewest sequence changes relative to the consensus. A clonecontaining the consensus was then generated by restrictionenzyme-mediated excision of non-consensus sequence and religation withfragments containing the consensus sequence, obtained by digestion ofthe other clones or by PCR amplification from HOI001 genomic DNA. Theconsensus sequence, including 1.5 kB upstream of the transcription startsite and approximately 300 base pairs downstream of the stop codon, islisted as SEQ ID NO: 1.

The GBSS gene from elite corn inbred line LH59 [SEQ ID NO: 7] wasisolated using the procedures and primers described above, and clonedinto the binary vector pMON68203. The resulting plasmid containing theLH59 GBSS is named pMON72510 (FIG. 5).

FIG. 1 shows the nucleic acid sequence alignment of the HOI001 GBSS [SEQID NO: 1] compared to the published sequence of Shure et al., supra [SEQID NO: 2] and the GBSS from LH59 [SEQ ID NO: 7], using the Omigasoftware package 2.0, (Accelrys Inc., San Diego, Calif.). The alignmentshows there are the following polymorphisms unique to the HOI001 GBSSsequence, that is not found in either the LH59 GBSS sequence or thepublished sequence of Shure et al., supra:

1. Single Nucleotide Polymorphisms:

-   -   a. T>C at position 158    -   b. G>A at position 337    -   c. C>A at position 343    -   d. C>A at position 349    -   e. G>A at position 441    -   f. C>T at position 666    -   g. G>C at position 777    -   h. T>A at position 878    -   i. C>T at position 980    -   j. T>A at position 1210    -   k. C>T at position 1216    -   l. A>T at position 1450    -   m. T>C at position 1709    -   n. A>G at position 1720    -   o. T>A at position 1721    -   p. G>C at position 1722    -   q. C>T at position 1761    -   r. G>A at position 1836    -   s. C>T at position 1852    -   t. G>A at position 1953    -   u. C>T at position 2043    -   v. C>T at position 2109    -   w. C>G at position 2110    -   x. G>C at position 2115    -   y. A>T at position 2448    -   z. C>T at position 2454    -   aa. T>G at position 2609    -   bb. A>G at position 2929    -   cc. G>T at position 2933    -   dd. C>T at position 2946    -   ee. G>T at position 3875    -   ff. T>A at position 4008    -   gg. T>C at position 4018    -   hh. T>G at position 4023    -   ii. C>A at position 4025    -   jj. C>T at position 4169    -   kk. A>T at position 4225    -   ll. C>A at position 4562

2. Insertions:

-   -   a. Sequence g at position 632    -   b. Sequence atgc at position 1185-1189    -   c. Sequence tgcaccagcagc at position 1456-1467    -   d. Sequence atgca at position 1746-1750    -   e. Sequence catcaca at position 1868-1874    -   f. Sequence ct at position 2100-2101    -   g. Sequence ccat at position 2488-2491    -   h. Sequence tat position 3810-3812

3. Deletions:

-   -   a. Sequence cgt at position 288-290    -   b. Sequence aa at position 704-705    -   c. Sequence c at position 882    -   d. Sequence atccg at position 1139-1143    -   e. Sequence ctctctg at position 1256-1262    -   f. Sequence tc at position 1714-1715    -   g. Sequence tgcaactgcaaatgca at position 1917-1932    -   h. Sequence g or a at position 3790    -   i. Sequence cgagccaggggt(t or c)gaaggcgaggagatcgcgccgctcgccaagg        agaacgtggccgcgccctgaagagttcggcct at position 4393-4467

FIG. 2 shows the alignment of the corresponding predicted amino acidsequences from the GBSS gene isolated from HOI001 [SEQ ID NO: 3], andthe GBSS gene described in Shure et al., supra [SEQ ID NO: 4],respectively. The results indicate that there is a sequence ofadditional amino acid residues on the carboxy terminus of the HOI001GBSS starting at approximately position 1441 and an area ofnon-alignment in the region of amino acid residue 55-60.

FIG. 3 shows the alignment of the corresponding predicted amino acidsequences from the Zea mays GBSS gene isolated from inbred LH59 [SEQ IDNO: 8], and the Zea mays granule bound starch synthase gene described inShure et al., supra, respectively. The results indicate that there is asequence of additional amino acid residues on the carboxy terminus ofthe HOI001 GBSS starting at approximately position 1441 and an area ofnon-alignment in the region of amino acid residue 55-60.

EXAMPLE 2

This example sets forth the construction of plant transformation vectorscontaining the sequences of the HOI001 GBSS and the GBSS from inbredline LH59, [SEQ ID NOs: 1 and 7, respectively].

The HOI001 GBSS sequence was cut from the consensus-corrected version ofpMON9480-2 using the restriction enzyme EcoR1. The resulting 4.7 kbfragment was purified following the manufacturer's protocol for theQiagen miniprep kit (Qiagen, Inc., Valencia, Calif.). The ends of thefragment were blunted following manufacturer's protocol in theStratagene PCR polishing kit (Stratagene, Inc., La Jolla, Calif.). Thefragment was then gel purified using the Qiagen Gel Extraction kit(Qiagen), and cloned into pMON68203, a binary vector for planttransformation. The binary vector, pMON68203, contains left and rightborders for T-DNA transfer, a CaMV 35S promoter::nptII::nos 3′ UTR plantselectable marker element (described in U.S. Pat. No. 6,255,560), andplant expression cassette sequences which include a 1.1 kb Z27 promoter(bp 19-1117 of Accession #S78780, Lopes et al., Mol. Gen. Genet.,247(5):603-613 (1995)) for endosperm expression, a corn hsp70 intron(base pairs 4-153 of the maize gene for heat shock 70 exon 2, Accession#X03679, Rochester et al., EMBO J., 5:451-458 (1986)), and a nos 3′ UTR,(base pairs 2924-2671 of the Agrobacterium tumefaciens strain C58 Tiplasmid, Accession #AE009420, Wood et al., Science, 294:2317-2323(2001)). The binary vector pMON68203 was digested with Stu1,dephosphorylated by incubating with shrimp alkaline phosphatase (RocheApplied Science, Indianapolis, Ind.) at 37° C. for 60 minutes andligated with the 4.7 kb gel purified fragment of the HOI001 GBSS,described above. The resulting plasmid was named pMON72506 (FIG. 4).

The GBSS from corn line LH59, [SEQ ID NO: 7], was similarly cloned intothe binary vector pMON68203, to form pMON72510.

EXAMPLE 3

This example describes the transformation of corn with the HOI001 GBSSand the GBSS from corn line LH59, using the vectors described in Example2.

The transformation vectors pMON72506 and pMON72510 were used totransform maize plants using the following procedure.

Corn plants are grown in a greenhouse under standard practices.Controlled pollinations were made. The ears of the plants are harvestedwhen the resulting hybrid embryos were 1.5 to 2.0 mm in length, usually10-15 days after pollination. After removing the husks, the kernels onthe ears were surface-sterilized by spraying with or soaking in 80%ethanol.

The Agrobacterium strain ABI, and an Agrobacterium tumefaciens binaryvector system were used for the transformations. Plasmids pMON72506 andpMON72510 were transformed into Agrobacterium tumefaciens according tomethods well known in the art. Prior to inoculation of corn cells theAgrobacterium cells are grown overnight at room temperature in AB medium(Chilton et al., Proc. Nat. Acad. Sci. U.S.A., 71:3672-3676 (1974))comprising appropriate antibiotics for plasmid maintenance and 200 μMacetosyringone. Immediately prior to inoculation the Agrobacterium cellswere pelleted by centrifugation, and resuspended in either CRN122 medium(2.2 g/L MS (Murashige and Skoog, Physiol. Plant, 15:473-497 (1962))basal salts, 2 mg/L glycine, 0.5 g/L niacin, 0.5 g/l L-pyridoxine-HCl,0.1 g/L thiamine, 115 mg/L L-proline, 36 g/L glucose, and 68.5 g/Lsucrose, pH 5.4) or CRN347 medium (CRN122 medium except with 0.44 g/L MSsalts, 10 g/L glucose, 20 g/L sucrose, and 100 mg/L ascorbic acid)containing 200 μM acetosyringone and 20 μM Ag NO₃.

The immature maize embryos were excised from individual kernels,immersed in an Agrobacterium suspension, and incubated at roomtemperature for 5-15 minutes. The Agrobacterium solution is thenremoved, and the inoculated immature embryos were transferredscutellum-side up from inoculation CRN122 medium to co-cultivationCRN123 medium (CRN122 medium except with 0.5 mg/L additionalthiamine-HCl, 20 g/L sucrose, 10 g/L glucose and 3 mg/L 2,4 D)containing 200 μM acetosyringone and 20 μM silver nitrate and incubatedat 23° C. for 1 day. Alternatively, excised embryos were cultured for8-11 days in 211V medium (3.98 g/L Chu N6 salts (Chu, C.C., The N6medium and its application to anther culture of cereal crops, in PlantTissue Culture Plant Tissue Culture. Proceedings of the PekinigSymposium, Boston, Mass. (1981), 43-50), 0.5 mg/L thiamine HCl, 0.5 mg/Lnicotinic acid; 1.0 mg/L 2,4 D, 20 g/L sucrose, 0.69 g/L L-proline, 0.91g/L L-asparagine monohydrate, 1.6 g/L MgCl₂ hexahydrate, 0.1 g/L caseinhydrolysate, 0.5 g/L MES, 0.1 g/L myo-inositol, and 16.9 mg/L silvernitrate, pH 5.8 solidified with 2 g/L Gelgro) and calli were inoculatedwith Agrobacterium CRN347 medium suspensions at 23° C. for 3 dayswithout adding additional media.

The embryos were then transferred to CRN220 selection medium (4.4 g/L MSsalts, 1.3 mg/L nicotinic acid, 0.25 mg/L pyridoxine HCl, 0.25 mg/Lthiamine HCl, 0.25 mg/L calcium pantothenate, 30 g/L sucrose, 12 mMproline, 0.05 g/L casamino acids, 500 mg/L carbenicillin, 200 mg/Lparomomycin, 2.2 mg/L picloram, 0.5 mg/L 2,4 D and 3.4 mg/L silvernitrate, pH 5.6 solidified with 7 g/L Phytagar), or calli aretransferred to CRN344 selection medium (3.98 g/L Chu N6 salts, 1.0 mg/Lthiamine HCl, 0.5 mg/L nicotinic acid; 1.0 mg/L 2,4 D, 20 g/L sucrose,0.69 g/L L-proline, 0.91 g/L L-asparagine monohydrate, 1.6 g/L MgCl₂hexahydrate, 0.1 g/L casein hydrolysate, 0.5 g/L MES, 0.1 g/Lmyo-inositol, 500 mg/L carbenicillin, 200 mg/L paromomycin and 16.9 mg/Lsilver nitrate, pH 5.8 solidified with 6 g/L Phytagar). After 2-3 weeksat 27° C. in the dark, surviving tissues were transferred to the sameselection medium and cultured for up to an additional 2 weeks ortransferred to regeneration medium as described below.

Plant regeneration is achieved by transferring the putative transgeniccallus from CRN220 to CRN232 medium (CRN220 medium lacking picloram,2,4-D, and silver nitrate, and containing 3.52 mg/L benzylaminopurine(BAP) and 250 mg/L carbenicillin) or from CRN344 medium to 217A medium(211RTTV lacking silver nitrate, 2,4 D, and paromomycin and containing3.52 mg/L BAP and 250 mg/L carbenicillin) and incubating for 5-7 days at27° C. Tissue is then transferred from CRN232 medium to CRN264 medium(4.4 g/L MS salts, 1.3 g/L nicotinic acid, 0.25 mg/L pyridoxine HCl,0.25 mg/L thiamine HCl, 0.25 mg/L calcium pantothenate, 10 g/L glucose,20 g/L maltose, 1 mM L-asparagine, 0.1 g/L myo-inositol, 250 mg/Lcarbenicillin and 100 mg/L paromomycin, pH 5.8 solidified with 6 g/LPhytagar) or from 217A medium to CRN346 medium (4.4 g/L MS salts, MSvitamins, 60 g/L sucrose, 0.05 g/L myo-inositol, 250 mg/L carbenicillin,75 mg/L paromomycin, pH 5.8 solidified with 6 g/L KOH) in Phytatrays,and incubated in the light at 28° C. until shoots with well-developedroots were produced (typically 2-3 weeks). These developing plantletswere then transferred to soil, hardened off in a growth chamber at 27°C., 80% humidity, and low light intensity for approximately 1 week, andthen transferred to a greenhouse and the R0 plants were grown understandard greenhouse conditions. The R0 plants were reciprocally crossedand both immature/developing kernels and mature kernels were collectedfrom each of the resulting plants for subsequent analyses. The resultsof the analyses are described below in Example 6.

These developing plantlets were then transferred to soil, hardened offin a growth chamber at 27° C., 80% humidity, and low light intensity forapproximately 1 week, and then transferred to a greenhouse. The R0plants were then grown under standard greenhouse conditions. Fertile R0plants were crossed to a non-transgenic recurrent inbred, with the R0plant serving as either the female or male (or occasionally both) in thecross. Both developing and mature F1 kernels were collected andanalyzed, from each of the resulting ears as described in Example 4. Theresults of the analyses are reported below in Example 5.

EXAMPLE 4

This example provides the analytical procedures to determine oil,protein, and starch levels in kernels from transgenic plants containingthe HOI001 GBSS gene or the LH59 GBSS gene.

Oil Content Analysis: Oil levels (on a mass basis and as a percent oftissue weight) of first generation single corn kernels and dissectedgerm and endosperm are determined by low-resolution ¹H nuclear magneticresonance (NMR) (Tiwari et al., JAOCS, 51:104-109 (1974); or Rubel,JAOCS, 71:1057-1062 (1994)), whereby NMR relaxation times of singlekernel samples are measured, and oil levels are calculated based onregression analysis using a standard curve generated from analysis ofcorn kernels with varying oil levels as determined gravimetricallyfollowing accelerated solvent extraction.

To compare oil analyses of transgenic and non-transgenic kernels, thepresence or absence of the transgene is determined by detection (or lackthereof) of a transgene-specific 517 bp PCR product, using a sequencewithin the Hsp70 intron as a 5′ primer, and a sequence within the HOI001GBSS gene as a 3′ primer: 5′ primer (primer number 19056): [SEQ ID NO:16] 5′-ATCTTGCTCGATGCCTTCTC-3′, 3′ primer (primer number 18986): [SEQ IDNO: 17] 5′-GCCTTCGCTTGTCGTGGGT-3′.

Oil levels in advanced generation seed are determined by NITspectroscopy, whereby NIT spectra of pooled seed samples harvested fromindividual plants are measured, and oil levels are calculated based onregression analysis using a standard curve generated from analysis ofcorn kernels with varying oil levels, as determined gravimetricallyfollowing accelerated solvent extraction or elemental (% N) analysis,respectively.

One-way analysis of variance and the Student's T-test are performed toidentify significant differences in oil (% kernel weight) between seedfrom marker positive and marker negative plants.

Alternatively, oil levels of pooled kernels from single ears aredetermined by low-resolution ¹H nuclear magnetic resonance (NMR) (Tiwariet al., JAOCS, 51:104-109 (1974); or Rubel, JAOCS, 71:1057-1062 (1994)),whereby NNM relaxation times of pools of kernels are measured, and oillevels are calculated based on regression analysis using a standardcurve generated from analysis of corn kernels with varying oil levels asdetermined gravimetrically following accelerated solvent extraction.

Protein Analyses: For kernel protein analysis, small bulk samplesconsisting of 50-100 kernels for each treatment are measured using nearinfrared reflectance spectroscopy (InfraTec model 1221, Teccator,Hogannas Sweden). This procedure is based upon the observation that alinear relation exists between the absorption of near infrared radiationand the quantity of chemical constituents comprised in a typical grainsample. Prior to analyzing unknown samples, spectral data is collectedwith calibration samples that are subsequently analyzed using a nitrogencombustion analysis technique (Murray, I., and P. C. Williams, 1987,Chemical Principles of Near-infrared Technology, In Near-InfraredTechnology in the Agricultural and Food Industries, P. Williams and K.Norris eds.). A multivariate model is developed using the spectral datafrom the spectrometer and the primary data. In the present case a PLS-1(Partial Least Squares Regression Type I) multivariate model isconstructed using 152 calibration samples. Each unknown sample isscanned on the spectrometer at least 5 times and its protein contentpredicted with each scan. Each time the sample is scanned it is addedback to the sample cuvette to minimize multiplicative scatteringeffects, which are not correlated to chemical property of interest. Thepredicted starch is averaged for the multiple scans and then reportedfor each sample.

Starch analyses: For kernel starch analysis, small bulk samplesconsisting of 50-100 kernels for each treatment are measured using nearinfrared reflectance spectroscopy (InfraTec model 1221, Teccator,Hogannas Sweden). This procedure is based upon the observation that alinear relation exists between the absorption of near infrared radiationand the quantity of chemical constituents comprised in a typical grainsample. Prior to analyzing unknown samples, spectral data is collectedwith calibration samples that are subsequently analyzed using standardwet chemistry analytical techniques (Murray, I., and P. C. Williams,1987, Chemical Principles of Near-infrared Technology, In Near-InfraredTechnology in the Agricultural and Food Industries, P. Williams and K.Norris eds.). A multivariate model is developed using the spectral datafrom the spectrometer and the primary data. Each unknown sample isscanned on the spectrometer at least 5 times and its starch contentpredicted with each scan. Each time the sample is scanned it is addedback to the sample cuvette to minimize multiplicative scatteringeffects, which are not correlated to chemical property of interest. Thepredicted starch is averaged for the multiple scans and then reportedfor each sample.

EXAMPLE 5

This example describes the analysis of kernels from plants transformedwith the HOI001 GBSS and the GBSS from LH59.

Kernels from a total of 54 transgenic events expressing the HOI001 GBSStransgenic allele were analyzed using the procedures set forth inExample 4. Table 1 shows whole kernel oil levels of transgenic(positive) and nontransgenic (negative) F1 kernels from ears of 20transgenic events analyzed by the single kernel NMR procedure describedin Example 4. Only results from events with a statistically significantincrease in oil (p<0.05) are shown.

The results demonstrate that transgenic kernels from ears of 20 of the54 events had statistically significant increases in whole kernel oilcontent (% dry weight) relative to nontransgenic kernels on the sameear. No events had a statistically significant decrease in oil. TABLE 1Positive Negative Pedigree n Mean n Mean Delta Prob > F ZM_S67336/LH17212 4.22 12 3.19 1.03 0.0000 ZM_S66829/LH172 8 4.33 16 3.44 0.89 0.0013ZM_S67359/LH172 12 4.36 12 3.52 0.84 0.0003 ZM_S71593/LH172 14 3.09 102.40 0.69 0.0258 ZM_S67345/LH172 8 3.31 15 2.67 0.64 0.0199ZM_S67335/LH172 9 3.85 15 3.25 0.61 0.0000 ZM_S71577/LH172 4 3.50 202.92 0.59 0.0298 ZM_S66804/LH172 10 3.50 14 2.94 0.56 0.0017ZM_S67348/LH172 6 3.76 16 3.20 0.56 0.0000 ZM_S67351/LH172 11 3.73 133.19 0.54 0.0173 ZM_S69437/LH172 9 3.70 15 3.16 0.54 0.0002ZM_S67331/LH172 13 3.70 11 3.17 0.53 0.0026 ZM_S67330/LH172 12 3.90 123.43 0.47 0.0071 LH172/ZM_S71581 17 3.47 7 3.07 0.40 0.0004ZM_S66805/LH172 11 3.76 13 3.37 0.39 0.0151 LH172/ZM_S69443 11 3.23 132.86 0.37 0.0243 LH172/ZM_S67360 11 3.34 13 2.98 0.36 0.0080LH172/ZM_S67338 17 3.01 7 2.68 0.34 0.0287 ZM_S71569/LH172 11 2.99 122.74 0.25 0.0354 LH172/ZM_S66817 14 3.05 9 2.83 0.22 0.0391

Transgenic kernels from R0 plants pollinated by non-transgenic inbredpollen (for example, pedigree ZM_S67336/LH172, positive) had both ahigher frequency of a significant oil increase (15/29 plants analyzed)and a higher average significant oil increase (0.61%) relative tokernels from non-transgenic inbred plants pollinated by transgenicpollen (for example, pedigree LH172/ZM_S66817, negative) from an R0 maleparent (5/37 plants analyzed, 0.34% significant oil increase). Theseresults suggest that the greater transgene dosage found in the endospermof kernels from the R0 plants, due to maternal inheritance effects, mayresult in a greater increase in oil.

Similarly, kernels from a total of 15 transgenic events containing theLH59 GBSS transgenic allele were analyzed. None of the kernels from earsof any of the events had statistically significant increases in wholekernel oil content (% dry weight) relative to nontransgenic kernels onthe same ear, indicating that the increase in oil was unique to theHOI001 GBSS allele.

EXAMPLE 6

This example describes the increase in oil levels obtained in transgenicF2 kernels from field-grown plants.

To ascertain the impact of the HOI001 GBSS gene on kernel oil levels offield-grown plants, 24-48 segregating F1 seed from each of 40 eventswere planted in a field nursery. Developing plants were screened for thepresence of the transgenic cassette by a non-lethal kanamycin resistanceassay, whereby an antibiotic solution (0.1% (w/v) kanamycin and 0.1%(w/v) paromomycin) is applied to the leaf surface and scored for thepresence (nontransgenic) or absence (transgenic) of necrotic lesions 1week after antibiotic application. Kernels were isolated from the earsof both transgenic plants and non-transgenic plants, and then wereassayed for kernel oil, protein, and starch by Near-InfraredTransmittance Spectroscopy.

Table 2 shows the mean whole kernel oil levels and the increase in wholekernel oil levels (Delta) in ears from plants containing (positive) andlacking (negative) the transgenic cassette containing the selectablemarker and the HOI001 GBSS gene. Oil levels were determined by the NITprocedure described in Example 4, and only events with a statisticallysignificant increase in oil (p<0.05) are shown. TABLE 2 PositiveNegative Event n Mean n Mean Delta Prob > F ZM_S67359 8 4.76 7 3.83 0.93<.0001 ZM_S71546 5 5.42 3 4.50 0.92 0.0012 ZM_S67354 2 4.85 13 4.02 0.83<.0001 ZM_S66817 3 4.37 1 3.70 0.67 0.0099 ZM_S71577 3 4.60 8 3.95 0.65<.0001 ZM_S67343 5 4.42 4 3.78 0.65 0.0142 ZM_S71555 5 5.10 3 4.47 0.630.0343 ZM_S71551 9 4.69 6 4.07 0.62 0.041 ZM_S69437 3 4.57 8 3.95 0.620.0002 ZM_S66804 7 5.04 7 4.44 0.60 0.0016 ZM_S67338 7 4.30 6 3.73 0.570.0068 ZM_S71573 3 4.87 3 4.40 0.47 0.0405 ZM_S67331 4 4.35 12 3.92 0.430.0025 ZM_S71594 2 4.35 8 3.95 0.40 0.0037 ZM_S67340 12 4.04 11 3.720.32 0.0115 ZM_S66800 1 4.30 8 3.95 0.30 0.0398

The results show whole kernel oil level was increased in transgenic earsrelative to nontransgenic ears (p<0.05) in 16 out of 36 events analyzed.

Table 3 shows the mean kernel starch levels (%) and the change in kernelstarch levels in ears from plants containing (positive) and lacking(negative) the transgenic cassette containing the selectable marker andthe HOI001 GBSS gene. Only events with a statistically significantincrease in oil (p<0.05) are shown.

Table 4 shows mean kernel protein levels (%) and the change in kernelprotein levels in ears from plants containing (positive) and lacking(negative) the transgenic cassette containing the selectable marker andthe HOI001 GBSS gene. Only events with a statistically significantincrease in oil (p<0.05) are shown.

Based on NIT analysis, starch levels in events with increases in oilwere lowered slightly (Table 3), and protein levels were mostlyunchanged (Table 4). TABLE 3 Positive Negative Event n Mean n Mean DeltaProb > F ZM_S67359 8 69.15 7 70.94 −1.79 0.0004 ZM_S71546 5 70.10 371.33 −1.23 0.0451 ZM_S67354 2 69.50 13 71.12 −1.62 0.0024 ZM_S66817 369.73 1 70.00 −0.27 0.5286 ZM_S71577 3 71.47 8 71.40 0.07 0.8702ZM_S67343 5 69.92 4 71.20 −1.28 0.0187 ZM_S71555 5 70.30 3 71.23 −0.930.118 ZM_S71551 9 70.49 6 71.23 −0.74 0.117 ZM_S69437 3 69.77 8 71.40−1.63 0.0018 ZM_S66804 7 71.19 7 72.27 −1.09 0.0073 ZM_S67338 7 69.81 671.25 −1.44 0.0009 ZM_S71573 3 69.77 3 70.67 −0.90 0.1352 ZM_S67331 470.10 12 71.23 −1.13 0.0026 ZM_S71594 2 70.75 8 71.40 −0.65 0.1906ZM_S67340 12 70.82 11 71.35 −0.53 0.0257 ZM_S66800 1 70.50 8 71.40 −0.900.16

TABLE 4 Positive Negative Event n Mean n Mean Delta Prob > F ZM_S67359 811.84 7 11.29 0.55 0.2942 ZM_S71546 5 12.86 3 12.30 0.56 0.1775ZM_S67354 2 12.55 13 12.07 0.48 0.2885 ZM_S66817 3 12.70 1 14.40 −1.700.1336 ZM_S71577 3 9.50 8 12.03 −2.53 0.0005 ZM_S67343 5 11.46 4 11.400.06 0.929 ZM_S71555 5 12.58 3 12.43 0.15 0.6995 ZM_S71551 9 12.23 611.97 0.27 0.4938 ZM_S69437 3 11.80 8 12.03 −0.23 0.7384 ZM_S66804 712.14 7 11.07 1.07 0.0304 ZM_S67338 7 12.26 6 11.87 0.39 0.452 ZM_S715733 11.37 3 11.30 0.07 0.8416 ZM_S67331 4 12.18 12 12.23 −0.05 0.9133ZM_S71594 2 11.80 8 12.03 −0.23 0.6682 ZM_S67340 12 11.44 11 11.28 0.160.578 ZM_S66800 1 11.40 8 12.03 −0.63 0.412

EXAMPLE 7

This example describes the increase in oil levels obtained in transgenicF2 hybrid kernels from field-grown plants.

To ascertain the impact of the HOI001 GBSS gene on kernel oil levels ofhybrid field-grown plants, 24-48 segregating F1 seed from each of 14events, having sufficient seed, were planted in a field nursery.Developing plants were screened for the presence of the transgeniccassette by the non-lethal Kanamycin resistance assay, described abovein Example 6. Pollen from transgenic plants was used to pollinate thestiff-stalk inbred LH244. The segregating F1 transgenic seed generatedwas then planted and the resultant plants were screened for the presenceof the transgene by the non-lethal Kanamycin resistance assay. F2 hybridkernels were isolated from ears from transgenic plants andnon-transgenic plants, and assayed for kernel oil by Nuclear MagneticResonance Spectroscopy, as described in Example 4.

Table 5 shows mean whole kernel oil levels and the increase (Delta) inwhole kernel oil levels in ears from hybrid plants containing (positive)and lacking (negative) the transgenic cassette containing the selectablemarker and the HOI001 GBSS gene. Oil levels were determined by the bulkset NMR procedure described in Example 4, and only events with astatistically significant increase in oil (p<0.05) are shown. The dataindicate that whole kernel oil level was increased in transgenic earsrelative to nontransgenic ears (p<0.05) in 9 out of 14 events analyzed.TABLE 5 Positive Negative Event n Mean n Mean Delta Prob > F ZM_S67354 64.43 1 3.00 1.43 0.0431 ZM_S67346 9 3.90 8 3.13 0.78 0.0003 ZM_S71546 94.24 5 3.58 0.66 0.0016 ZM_S71556 10 4.12 4 3.48 0.65 0.0044 ZM_S71577 83.94 6 3.30 0.64 0.0047 ZM_S71594 10 3.91 7 3.30 0.61 0.0041 ZM_S7157310 4.02 6 3.42 0.60 0.0001 ZM_S67343 10 3.72 4 3.15 0.57 0.0009ZM_S67331 8 3.80 3 3.40 0.40 0.0281

EXAMPLE 8

This example describes the elevation of GBSS activity in corn endospermtissue expressing the HOI001 GBSS gene.

Developing ears from F1 plants screened for the presence of thetransgenic cassette by the non-lethal Kanamycin resistance assay wereharvested and immediately frozen at 24 days post pollination.Segregating F2 kernels were removed from the ear, then dissected intogerm and endosperm fractions. Individual dissected kernels wereidentified as transgenic or nontransgenic by screening for the abilityto PCR-amplify a portion of the transgenic cassette from genomic DNAisolated from individual germs using transgene-specific primers, asdescribed in Example 4. For each of six events, approximately 10endosperms from the corresponding transgenic and nontransgenic kernelswere pooled separately.

Each endosperm pool was ground to a fine powder with a mortar and pestleunder liquid nitrogen, and starch granules were isolated in triplicateaccording to the procedure of Shure et al., Cell, 35(1):225-233 (1983).Granule-bound starch synthase activity was assayed on the isolatedgranules using the method of Vos-Scheperkeuter et al., Plant Physiol.,82:411-416(1986).

Table 6 shows the granule-bound starch synthase activity (pmol/min/mgstarch) in developing F2 endosperm containing or lacking the HOI001 GBSStransgenic cassette. Values shown are means and standard errors oftriplicate assays. The data indicates that starch granules fromtransgenic kernels generally had elevated GBSS activity, indicating thatthe effect of the HOI001 allele on oil levels is not a function ofreducing overall GBSS activity, but functions by the addition of anactivity uniquely encoded by the HOI001 GBSS gene. TABLE 6 Trans- Non-genic transgenic Event Mean SE mean SE p > F S67338 585 41 455 15 0.0392S67359 583 20 521 15 0.0665 S71546 635 16 540 23 0.0281 S66804 587 50454 20 0.0702 S71551 588 9 574 11 0.3712 S71555 486 24 464 12 0.4641

EXAMPLE 9

This example describes the isolation and sequencing of the coding regionof the GBSS cDNA from corn line HOI001.

mRNA was extracted from developing corn endosperm tissue from HOI001, 22days after pollination, using a procedure adapted from Opsahl-Ferstad etal., Plant J., 12(10):235-246 (1997). Briefly, developing endosperm from3 separate kernels was pooled, frozen in liquid nitrogen, and thenpulverized with a mortar and pestle. Approximately 50 mg frozen powderedendosperm was extracted with 0.5 mL buffer (0.5 M LiCl, 10 mM EDTA, 5 mMdithiothreitol, 100 mM Tris-HCl, pH 8.0, 1% (w/v) SDS). This aqueousextract was then extracted with phenol:chloroform:isoamyl alcohol(25:24:21), and the organic fraction was discarded. Nucleic acids wereprecipitated from the aqueous fraction by addition of an equal volume ofisopropyl alcohol followed by centrifugation. The resulting supernatantwas discarded. The pellet containing the mRNA was washed twice with 70%ethanol, dried, and then resuspended in 50 μL water containing 0.1%(v/v) diethylpyrocarbonate.

First-strand cDNA was synthesized from the isolated mRNA using theClontech SMART· cDNA synthesis system (BD Biosciences). Thisfirst-strand cDNA was used as template to amplify HOI001 GBSS cDNAsequences using primers containing an EcoRV restriction site followed by18 bp of the predicted translational start site (5′ primer) and aSse83871 restriction site followed by 17 bp of the predicted 3′ end upto the translation stop site (3′ primer): 5′ Primer (primer number20095): [SEQ ID NO: 9] 5′-GGATATCACCATGGCGGCTCTGGCCACG-3′, 3′ Primer(primer number 20092): [SEQ ID NO: 10]5′-GTCCTGCAGGCTACACATACTTGTCCA-3′.

The resulting 1.8 kB amplification products from independentamplification reactions were isolated by agarose gel electrophoresis,cloned independently into the PCR 2.1 cloning vector using the TOPO TAcloning kit (Invitrogen), and then transformed into an E. coli host.Multiple colonies were isolated from each transformation, plasmid DNAwas prepared from cultures grown from each colony, and then the insertin each plasmid preparation was sequenced. Alignment of these sequencesgenerated a consensus sequence containing an open reading frameequivalent to that predicted to be encoded by the HOI001 GBSS gene,although no specific insert sequence was equivalent to the consensus.One clone (designated 7345705-10) had an insert sequence with singlebase pair deletion relative to the consensus. This clone was used togenerate a plasmid (designated pMON81463) containing the consensussequence by inserting the additional nucleotide using the Quick-Changemutagenesis kit (Stratagene). This sequence, representing the codingregion of the HOI001 GBSS cDNA, is listed in SEQ ID NO: 11.

EXAMPLE 10

This example sets forth the construction of plant transformation vectorscontaining the HOI001 GBSS cDNA coding region [SEQ ID NO: 11], designedto obtain different levels, timing and spatial patterns of expression,and the subsequent transformation of corn.

A plant transformation vector containing the HOI001 GBSS coding regiondriven by a Z27 promoter was constructed. The HOI001 GBSS coding regionwas isolated from pMON81463 by restriction digest with EcoRV andSse83871, and cloned into the binary vector pMON71274. This binaryvector contains left and right borders for T DNA transfer; a rice Actinpromoter::rice Actin intron::CP4::nos 3′ UTR, plant selectable markerelement; and plant expression cassette sequences which include a 1.1 kbZ27 promoter (bp 19-1117 of Genbank Accession #S78780, Lopes et al.,Mol. Gen. Genet., 247(5):603 613 (1995)) for endosperm expression; acorn hsp70 intron (base pairs 4-153 of the maize gene for heat shock 70exon 2, Genbank Accession #X03679, Rochester et al., EMBO J., 5:451-458(1986)), and a globulin 3′ UTR. The resulting plasmid was namedpMON81464 (FIG. 7).

A second plant expression binary vector containing the wheat highmolecular weight glutenin promoter (bp 2647-3895 of Genbank AccessionX12928, version X12928.3, originally described in Anderson et al.,Nucleic Acids Res., 17:461-462 (1989)) and the corn hsp70 intron, fusedto the GBSS coding region, fused to the wheat HSP17n3′ UTR (bp532-741 ofGen Bank Accession X13431, version X13431.1, McElvain and Spiker,Nucleic Acids Res., 17:1764 (1989)), was constructed. A sequencecontaining the wheat high molecular weight glutenin promoter fused tothe corn hsp70 intron was amplified from an intermediary vector using 5′and 3′ primers containing AscI and NotI restriction sites, respectively:5′ Primer (primer number 21084): [SEQ ID NO: 12]5′-GGCGCGCCGTCGACGGTATCGATAAGCTTGC-3′, 3′ Primer (primer number 21085):[SEQ ID NO: 13] 5′-GCGGCCGCCCGCTTGGTATCTGCATTACAATG-3′.

The amplification product, containing the promoter and intron fragmentwith the introduced restriction sites, was purified by agarose gelelectrophoresis and cloned into pCR2.1 TOPO (Invitrogen) to generate aplasmid vector for E. coli transformation (pOP28). After transformationinto an E. coli vector, plasmid DNA was isolated, digested with AscI andNotI, and the purified fragment was cloned into the binary vectorpMON71274 to generate a vector (pOP29) containing a cassette with thewheat high molecular weight glutenin promoter fused to the corn HSP70intron fused to the globulin 3′ UTR. The HOI001 GBSS coding region wasisolated by digestion of pMON81464 with NotI/Sse83871 and cloned intopOP29 to generate the binary vector pOP31 containing an expressioncassette with the wheat high molecular weight glutenin promoter fused tothe corn HSP70 intron fused to the HOI001 GBSS coding region fused tothe globulin 3′ UTR. The promoter/intron/HOI001 GBSS coding regionfragment was then isolated from pOP31 by digestion with AscI/Sse83871and then cloned into the plant binary vector pMON71290 containing a geneof interest cassette with the TR7 3′ UTR to generate pOP35, containingan expression cassette with the wheat high molecular weight gluteninpromoter fused to the corn HSP70 intron fused to the HOI001 GBSS codingregion fused to the TR7 3′ UTR. The promoter/intron/HOI001 GBSS codingregion fragment was then isolated from pOP35 by digestion withAscI/Sse83871 and then cloned into the plant binary vector pMON67647,containing a gene of interest cassette with the wheat HSP17 3′ UTR. Theresulting plasmid contained an expression cassette with the wheat highmolecular weight glutenin promoter fused to the corn HSP70 intron fusedto the HOI001 GBSS coding region fused to the wheat HSP17 3′ UTR. Thisplasmid, was named pMON68298, is shown in FIG. 8.

A third plant expression binary vector containing the promoter and 5′UTR of the HOI001 GBSS gene fused to the HOI001 GBSS coding region,fused to the corn globulin 3′ UTR, was constructed. The HOI001 GBSSpromoter and 5′ UTR (which also contained the first predicted intron)was isolated by PCR amplification from pMON72506, using a 5′ primer thatcontains the restriction site for PmeI: 5′ Primer (primer number 20362):[SEQ ID NO: 14] 5′-GATCGTTTAAACGTTCGTGTGGCAGATTCATC-3′, 3′ Primer(primer number 20363): [SEQ ID NO: 15]5′-GACGTGGCCAGAGCCGCCATGCCGATTAATCCACTGCATAG-3′.

The amplification product, a fragment containing 1125 bp upstream of thepredicted HOI001 GBSS translational start site and 20 bp of thepredicted coding sequence from pMON72506 (corresponding to bp 17-1162 ofSEQ ID NO: 1), was purified by agarose gel electrophoresis and clonedinto pCR2.1 TOPO (Invitrogen) to generate pMON81466.

The HOI001 GBSS coding region was removed from pMON81463 and cloned intothe vector pMON81466 to generate pMON81468, containing the HOI001 GBSSpromoter/5′ UTR fused to the HOI001 GBSS coding region, with 45 bpextraneous polylinker sequence between the promoter/UTR and codingregion elements. This extraneous sequence was then deleted by digestionof pMON81468 with MluI to remove a 780 bp fragment spanning theextraneous sequence, then reannealing with the analogous 735 bp fragment(lacking the extraneous sequence), generating pMON81469. This 735 bpfragment was generated by digestion of pMON72506 with MluI and isolatingthe resulting fragment. This entire promoter/UTR/coding region sequencewas then isolated from pMON81469 by digestion with PmeI and Sse83871,and cloned into the binary vector pMON71274 to generate the binaryvector pMON81465. This vector contained an expression cassette with thepromoter and 5′ UTR of the HOI001 GBSS gene fused to the GBSS codingregion fused to the corn globulin 3′ UTR (FIG. 9).

These three plant transformation vectors are transformed into an elitecorn inbred (LH244) (Corn States Hybrid Serv., LLC, Des Moines, Iowa).Briefly, ears containing immature embryos are harvested approximately 10days after pollination and kept refrigerated at 4° C. until use (up to 5days post-harvest). The preferred embryo size for this method oftransformation is ˜1.0-2.0 mm. This size is usually achieved 10 daysafter pollination inside the greenhouse with the growth conditions of anaverage temperature of 87° F., day length of 14 hours with supplementallighting supplied by GE 1000 Watt High Pressure Sodium lamps.

Immature embryos are isolated from surface sterilized ears and directlydropped into the prepared Agrobacterium cell suspension in a 1.5-mLmicrocentrifuge tube. The isolation lasts continuously for 15 minutes.The tube is then set aside for 5 minutes, resulting in a totalinoculation time for individual embryos from 5 to 20 minutes. After theAgrobacterium cell suspension is removed using a fine tipped steriletransfer pipette, the immature embryos are transferred onto a co-culturemedium (Table 7). The embryos are then placed on the medium with thescutellum side facing up. The embryos are cultured in a dark incubator(23° C.) for approximately 24 hours.

The embryos are then transferred onto a modified MS medium (MSW50, Table7) supplemented with 0.1 or 0.25 mM glyphosate and 250 mg/Lcarbenicillin to inhibit Agrobacterium in Petri dishes (100 mm×25 mm).The cultures are incubated in a dark culture room at 27° C. for 2-3weeks. All the callus pieces are then transferred individually onto thefirst regeneration medium (MS/6BA, Table 7) supplemented with the samelevels of glyphosate. The cultures are grown on this medium and in aculture room with 16 hours light/8 hours dark photoperiod and 27° C. for5-7 days. They are then transferred onto the second 15 regenerationmedium (MSOD, Table 7) in Petri dishes (100 mm×25 mm) for approximately2 weeks. All the callus pieces with regenerating shoots and livingtissue are transferred onto the same medium contained in phytatrays forshoots to grow further prior to being transferred to soil (approximately2-4 weeks). The regeneration media (MS6BA and MSOD) are all supplementedwith 250 mg/L carbenicillin and 0.1 or 0.25 mM glyphosate.

These developing plantlets are then transferred to soil, hardened off ina growth chamber at 27° C., 80% humidity, and low light intensity forapproximately 1 week, and then transferred to a greenhouse and grownunder standard greenhouse conditions. The resulting kernels arecollected and analyzed as described in Example 4. The results indicatethat the different promoters have different impacts on oil accumulationbased upon the strength and timing of the expression of the HOI001 GBSScoding region. TABLE 7 ,Composition of media used in corntransformation. Co-culture Component Media MSW50 MS/6BA MSOD MS salts2.2 g/L 4.4 g/L 4.4 g/L 4.4 g/L Sucrose 20 g/L 30 g/L 30 g/L Maltose 20g/L Glucose 10 g/L 10 g/L 1-Proline 115 mg/L 1.38 g/L 1.36 g/L Casamino500 mg/L 50 mg/L Acids Glycine 2 mg/L 2 mg/L 1-Asparagine 150 mg/LMyo-inositol 100 mg/L 100 mg/L 100 mg/L Nicotinic acid 0.5 mg/L 0.5 mg/L1.3 mg/L 1.3 mg/L Pyridoxin HCl 0.5 mg/L 0.5 mg/L 0.25 mg/L 0.25 mg/LThiamine-HCl 0.5 mg/L 0.6 mg/L 0.25 mg/L 0.25 mg/L Ca 0.25 mg/L 0.25mg/L Pantothenate 2,4-D 3 mg/L 0.5 mg/L Picloram Silver Nitrate 1.7 mg/LBAP 3.5 mg/L

Co-culture medium was solidified with 5.5 mg/l low EEO agarose. Allother media were solidified with 7 g/l Phytagar for NPTII selection andwith 3 g/l phytagel for glyphosate selection.

EXAMPLE 11

This example sets forth the use of the polymorphisms in the HOI001 GBSSgene as molecular markers to accelerate incorporation of HOI001 GBSSsequence polymorphisms into other corn germplasm with the result ofincreasing oil in the kernel.

The present invention provides a corn plant with increased kernel oilselected for by use of marker assisted breeding wherein a population ofplants are selected for the presence of a polymorphism sequence uniqueto the HOI001 GBSS gene (SEQ ID NO: 1). Example 1, above, listspolymorphisms unique to the HOI001 GBSS sequence, that is not found ineither the LH59 GBSS sequence or the published sequence (Shure et al.,supra).

The selection of plants having the HOI001 GBSS gene for high oilcomprises probing genomic DNA of the resulting plants, through theselection process, for the presence of the molecular marker for theHOI001 GBSS gene. The molecular marker is a DNA molecule representing aunique polymorphism in the HOI001 GBSS gene that functions as a probe orprimer to a target HOI001 GBSS in a plant genome. The selectedpolymorphism may or may not be from a coding region of the gene. Theplants containing the HOI001 GBSS gene are continued in the breeding andselection process.

1. A substantially purified nucleic acid molecule selected from thegroup consisting of: a) a nucleic acid molecule comprising SEQ ID NO: 1or the complement thereof; b) a nucleic acid molecule comprising SEQ IDNO: 11 or the complement thereof; and c) a nucleic acid molecule whichencodes a polypeptide having at least about 94% amino acid identity withSEQ ID NO:
 3. 2. An expression cassette comprising a nucleic acidmolecule of claim 1, wherein said nucleic acid molecule is operablylinked to a promoter, which is functional in a plant cell.
 3. A plantcell comprising the expression cassette of claim
 2. 4. A plantcomprising the plant cell of claim
 3. 5. A plant according to claim 4,wherein the plant is a monocot.
 6. A plant according to claim 5, whereinthe monocot plant is corn.
 7. Seeds obtained from a corn plant, whereinthe seeds comprise the nucleic acid molecule of claim
 1. 8. An animalfeed obtained from the seeds of claim
 7. 9. A plant having stablyincorporated into it's genome a nucleic acid molecule selected from thegroup consisting of: a) a nucleic acid molecule comprising SEQ ID NO: 1or the complement thereof; b) a nucleic acid molecule comprising SEQ IDNO: 11 or the complement thereof; and c) a nucleic acid molecule whichencodes a polypeptide having at least about 94% amino acid identity withSEQ ID NO:
 3. 10. A plant according to claim 9, wherein the plant is amonocot.
 11. The monocot plant of claim 10, wherein the plant is corn.12. Seeds obtained from the plant of claim
 11. 13. Oil obtained from theseeds of claim
 12. 14. An animal feed obtained from the seeds of claim12.
 15. A method of producing a plant having increased levels of oilproduction, wherein the method comprises: (a) transforming a plant withan expression cassette comprising a nucleic acid molecule selected fromthe group consisting of: i) a nucleic acid molecule comprising SEQ IDNO: 1 or the complement thereof; ii) a nucleic acid molecule comprisingSEQ ID NO: 11 or the complement thereof; and iii) a nucleic acidmolecule which encodes a polypeptide having at least about 94% aminoacid identity with SEQ ID NO: 3; wherein said expression cassettefurther comprises a promoter region functional in a plant cell, operablylinked to said nucleic acid molecule; and (b) growing the transformedplant.
 16. The method claim 15, wherein the plant is a monocot.
 17. Themethod of claim 16, wherein the monocot plant is corn.
 18. The method ofclaim 17, wherein the promoter region is an endosperm promoter region.19. The method of claim 18, wherein the promoter region is the Z27promoter.
 20. A method of selecting corn germplasm, comprising the stepsof: a) identifying at least one polymorphism unique to the HOI001 GBSSsequence represented in SEQ ID NO: 1; b) selecting a fragment of SEQ IDNO: 1 containing at least part of one of the identified polymorphisms tobe used as a molecular marker; c) assaying corn plants for the presenceof the marker; and d) selecting plants that contain the marker.